Peptide-based proteasome inhibitors for treating conditions mediated by senescent cells and for treating cancer

ABSTRACT

The proteasome inhibitors of this invention include peptide-based compounds with a short linear sequence of amino acids. An oxo or thio group is attached to the N-terminal amino acid. A protein-reactive electrophilic group such as an epoxyketone, an aziridinylketone, or a beta-lactone is attached to the C-terminal amino acid. Upon contact with a proteasome complex in a target cell, the electrophilic group reacts with a functional group in or near a binding pocket or active site of the proteasome, forming a covalent bond and thereby inactivating the proteasome. These and other proteasome inhibitors can be screened for binding affinity and an ability to selectively eliminate senescent cells or cancer cells. Compounds that selectively remove senescent cells can be developed for the treatment of conditions such as osteoarthritis, ophthalmic disease, pulmonary disease, and atherosclerosis.

PRIORITY

This application is a continuation-in-part of international applicationPCT/US2018/068190, filed Dec. 31, 2018, which claims the prioritybenefit of U.S. provisional patent applications 62/612,411, 62/612,414,62/612,416, 62/612,417, and 62/612,418, all filed Dec. 30, 2017,provisional application 62/676,692, filed May 25, 2018, andinternational application PCT/US2018/068003, filed Dec. 28, 2018. Theaforelisted applications are all hereby incorporated herein by referencein their entirety for all purposes.

FIELD OF THE INVENTION

The technology disclosed and claimed below relates generally to theactivity of proteasomes and their inhibition in target cells. Inparticular, this disclosure provides a new family of proteasomeinhibitors that are suited for use in treating conditions meditated bysenescent cells and for treating cancer.

BACKGROUND

Senescent cells are characterized as cells that no longer havereplicative capacity, but remain in the tissue of origin, eliciting asenescence-associated secretory phenotype (SASP). It is a premise ofthis disclosure that many age-related conditions are mediated bysenescent cells, and that selective removal of the cells from tissues ator around the condition can be used clinically for the treatment of suchconditions.

US 2016/0339019 A1 (Laberge et al.) describes treatment of certainage-related conditions using MDM2 inhibitors, Bcl inhibitors, and Aktinhibitors. US 20170266211 A1 (David et al.) describes the use ofparticular Bcl inhibitors for treatment of age-related conditions. U.S.Pat. Nos. 8,691,184, 9,096,625, and 9,403,856 (Wang et al.) describe Bclinhibitors in a small-molecule library.

Other disclosures related to the role of senescent cells in humandisease include the pre-grant publications US 2017/0056421 A1 (Zhou etal.), WO 2016/185481 (Yeda Inst.), US 2017/0216286 A1 (Kirkland et al.),and US 2017/0281649 A1 (David); and the articles by Furhmann-Stroissnigget al. (Nat Commun. 2017 Sep. 4; 8(1):422), Blagosklonny (Cancer BiolTher. 2013 December; 14(12):1092-7), and Zhu et al. (Aging Cell. 2015August; 14(4):644-58).

In a previously unrelated field, the targeting of proteasome complexesto treat cancer and other conditions is referred to in Park et al.,Transl Res. 2018 August; 198:1-16, and in Dou et al., Curr Cancer DrugTargets. 2014; 14(6):517-36. Proteasome inhibitor patents since 2010 arereviewed by Metcalf et al., Expert Opin Ther Pat. 2014 April;24(4):369-8.

SUMMARY

The new proteasome inhibitors described below are peptide-basedcompounds with a short linear sequence of amino acids. An oxo or thiogroup is attached to the N-terminal amino acid. A protein-reactiveelectrophilic group such as an epoxyketone, an aziridinylketones, or abeta-lactone is attached to the C-terminal amino acid. Upon contact witha proteasome complex in a target cell, the electrophilic group reactswith a functional group in or near a binding pocket or active site ofthe proteasome, forming a covalent bond and thereby inactivating theproteasome.

Certain biochemical pathways are more active in senescent cells than inother cell types. Previous medicines for treating senescent conditionshave been based on inhibitors of the Bcl protein family, or MDM2. Thisdisclosure is based in part on the discovery that the proteasome pathwayis also selectively expressed in senescent cells. This provides a windowof opportunity for targeting senescent cells without unduly impairingthe activity of neighboring non-senescent cells in the target tissue.Contacting senescent cells in vitro or in vivo with small-moleculesenolytic agents selectively modulates or eliminates such cells. Theinhibitors can be used for administration to a target tissue in asubject, thereby selectively eliminating senescent cells in or aroundthe tissue, and relieving one or more symptoms or signs of disease oraging that are initiated or mediated by the senescent cells.

The new proteasome inhibitors described below, and proteasome inhibitorshaving other structures can be screened for binding affinity and anability to eliminate senescent cells or cancer cells selectively.Compounds with the requisite activity can be developed for the treatmentof conditions such as osteoarthritis, ophthalmic disease, pulmonarydisease, and atherosclerosis.

The invention is put forth in the description that follows, in thedrawings, and in the appended claims.

DRAWINGS

FIGS. 1A, 1B, and 1C show structures of exemplary proteasome inhibitorsaccording to this invention. FIG. 1D shows previously known proteasomeinhibitors that can be newly applied to the treatment of senescentdisease.

FIGS. 2A, 2B, and 2C show results from a screening assay to identifycompounds that selectively kill senescent cells, leaving non-senescentcells intact. FIG. 2A provides data for senolytic activity andproteasome binding for structures selected from FIGS. 1A and 1C. FIG. 2Bprovides data for senolytic activity and proteasome binding forstructures selected from FIG. 1B.

FIGS. 3A, 3B, and 3C show expression of senescent cell markers p16,IL-6, and MMP13 respectively in an osteoarthritis model. FIG. 4A showsthat an effective senolytic agent restores symmetrical weight bearing totreated mice in the osteoarthritis model. FIGS. 4B, 4C, and 4D areimages showing histopathology of the joints in these mice. The testsenolytic agent helps prevent or reverses destruction of theproteoglycan layer.

FIG. 4A shows that an effective senolytic agent restores symmetricalweight bearing to treated mice in the osteoarthritis model. FIGS. 4B,4C, and 4D are images showing histopathology of the joints in thesemice. The test senolytic agent helps prevent or reverses destruction ofthe proteoglycan layer.

FIGS. 5A and 5B show reversal of both neovascularization andvaso-obliteration in the mouse oxygen-induced retinopathy (OIR) modelwhen intravitreally administered with a senolytic agent.

FIGS. 5C and 5D are taken from the streptozotocin (STZ) model fordiabetic retinopathy. STZ-induced vascular leakage is attenuated withthe intravitreal administration of a senolytic agent.

FIG. 6 shows that removing senescent cells with a senolytic agent helpsrestore oxygen saturation (SPO₂) in a mouse model for cigarette smoke(CS) induced COPD (chronic obstructive pulmonary disease).

FIG. 7 shows data taken from a mouse model for atherosclerosis, in whichinbred mice lacking the LDL receptor were fed a high-fat diet. The rightpanel shows staining for plaques in the aorta. The middle panel showsquantitatively that the surface area of the aorta covered with plaqueswas reduced by treatment with a senolytic agent.

FIG. 8 is a schematic portrayal of the proteasome pathway and its rolein the destruction of proteins marked by ubiquitination.

DETAILED DESCRIPTION

Senescent cell medicine encompasses the paradigm that many conditionsthat are associated with aging or tissue damage are caused or mediatedby senescent cells. These are cells that no longer replicate, but have asecretory phenotype that includes secretion of factors that triggerpathophysiology.

This disclosure shows that the proteasome pathway is active in senescentcells, and can be used as an effective means for removing senescentcells from a target tissue, as an alternative to other targets. A newfamily of proteasome inhibitors are provided as part of the invention.

Proteasome Function

The proteasome is a protein complex consisting of 28 subunits arrangedin four stacked rings, each having 7 subunits (two outer α 1-7-rings andtwo inner β 1-7-rings). The catalytic protease activity derives from 3of the β subunits. The chymotrypsin-like (CT-L) activity (β5),trypsin-like (T-L) activity (β2), and caspase-like or post-acid (PA)activity (β1).

FIG. 8 provides a schematic depiction of the role of proteasomes incells. The proteasome is the effector component of theubiquitin-proteasome-system (UPS) where it degrades ubiquitinatedproteins by proteolysis. Ubiquitination is a post-translationmodification where the ubiquitin protein is covalently attached tolysine residues. A series of enzymes carries out a cascade of reactionsinvolving E1 activating, E2 conjugating and E3 ligating enzymes.Ubiquitin itself contains lysine residues which can serve to propagatethe cycle of ubiquitination with the addition of more ubiquitin units.Ubiquitination at K48 and K11 mark proteins for degradation by theproteasome. These ubiquitinated proteins marked for degradation consistof components of signaling pathways and misfolded or damaged proteins.

The UPS pathway is important for replenishing cells with amino acidsrequired for survival. Reduced levels of proteasome have been observedin senescent cells with corresponding increases in levels of bothdamaged (oxidized) and ubiquitinated proteins. Several proteins involvedin survival and apoptotic pathways are regulated via the UPS system.Senescent cells have a dysregulated survival/apoptosis balance,proteasome inhibition is proposed to be senolytic.

New Proteasome Inhibitors

FIGS. 1A, 1B, and 1C depict a family of small molecule compounds thatwere synthesized for the first time as illustrations of this invention.These compounds and their analogs are designed for inhibiting proteasomeactivity, and are suitable for testing and development for the purposeof eliminating senescent cells or treating senescence-associatedconditions. They can also be used for the purpose of eliminatingcancerous or malignant cells in the treatment of cancer.

Proteasome inhibitors included in this invention are peptidic compoundshaving a plurality of (typically 3 to 7) peptidic units arranged in asequence, where the first unit of the sequence has an oxo or thio groupat a first terminal and the last unit in the sequence has anelectrophilic group at a second terminal. The peptidic units of theproteasome inhibitors of this disclosure can include an amino acidresidue or a peptidomimetic unit that mimics the residue, or a componentof the residue. When the peptidic compounds are composed of amino acidresidues, the amino group of the N-terminal residue is replaced with anoxo or thio group and the C-terminal residue is modified to include anelectrophilic group.

An exemplary proteasome inhibitor has the structure shown in Formula(I):R⁰—X-(A)_(n)-Z  (I)where;

-   -   R⁰ is selected from H, alkyl, substituted alkyl, alkanoyl,        substituted alkanoyl, alkylaminocarbonyl, substituted        alkylaminocarbonyl, alkoxycarbonyl, substituted alkoxycarbonyl,        alkylaminothiocarbonyl, substituted alkylaminothiocarbonyl,        alkoxythiocarbonyl, substituted alkoxythiocarbonyl and        promoiety;    -   (A)_(n) is a sequence of n peptidic units A¹ to A^(n), each        independently selected from amino acid residues and        peptidomimetic units, where:        -   n is 2-7;        -   the A¹ unit includes a first terminal group X, wherein X is            O (i.e., oxo) or S (i.e., thio); and        -   the A^(n) unit includes a second terminal group Z that is a            proteasome-reactive electrophilic group.

When n is 4, the proteasome inhibitors of this disclosure may conform tothe structure shown in Formula (Ia):R⁰—X-A¹-A²-A³-A⁴-Z  (Ia).where the A₄ unit includes a modified terminal Z. The term modifiedterminal Z refers to a unit of the proteasome inhibitor that can be anamino acid residue or peptidomimetic residue where a C-terminalcarboxylic acid group that is found in a naturally occurring peptide isreplaced with proteasome-reactive electrophilic group Z.

Amino acid residues of the proteasome inhibitors of this disclosure canbe α-amino acids or β-amino acids, where the amino acids in this contextcan be naturally or non-naturally occurring, L-amino acids or D-aminoacids. Peptidomimetic units in this context can include small organicgroup designed to mimic an amino acid residue or a dipeptide residue, ora fragment or smaller component of such residues (e.g., a peptide bond).The peptidomimetic unit can be referred to as a bioisostere of an aminoacid or dipeptide residue or component thereof. An example of such acomponent is an amide bond bioisostere. Amide bond bioisosteres includegroups such as ester, ethylene, thioamide, alkylamino, alkylketone,alkylether, N-alkyl-amide, tetrazole and pyrrole. Peptidomimetic unitswhich can be utilized as bioisosteres in the inhibitors of thisdisclosure include hydroxyethylamine, hydroxyethylene,1,2-dihydroxyethylene, hydroxyamide, α-ketoamide, amino ketone andstatin dipeptide isosteres, and azapeptide, peptoid and retroinversounits. Other bioisosteres of α-amino acid residues includequinoxaline-2,4(1H)-dione, quinoxaline-2,3(1H)-dione andquinolin-2(1H)-one, azagrevellin, 3,4-diamino-3-cyclobutene-1,2-dione,and azepine-derived structures. In the proteasome inhibitors of thisdisclosure, one or more units of the sequence is a peptidomimetic unit.The A¹ unit of formula (I)-(Ia) can be referred to as a peptidomimeticunit because it includes an oxo (i.e., X=O) or thio (i.e., X=S) groupinstead of a N-terminal amine group of an amino acid residue. The A⁴unit of formula (Ia) can be referred to as a peptidomimetic unit becauseit includes a terminal group Z instead of a C-terminal carboxylic acidof a naturally occurring amino acid residue.

The proteasome inhibitors of this disclosure may also conform to thestructure shown in Formula (II):

where:

-   -   B¹ to B⁴ are each independently a branching group selected from        CH, CR″ and N, wherein R″ is C₍₁₋₆₎alkyl or substituted        C₍₁₋₆₎alkyl;    -   L¹ to L⁴ are each independently a linking group; and    -   R¹ to R⁴ are independently selected from alkyl, substituted        alkyl, aralkyl, substituted aralkyl, heteroarylalkyl and        substituted heteroarylalkyl.

The sequence of branching and linking groups shown in Formula (II) canprovide a peptidic backbone for the proteasome inhibitors of thisdisclosure to which amino acid sidechain groups of interest R¹ to R⁴ canbe connected in a desirable configuration. The branching groups B¹ to B⁴of the peptidic backbone can be trivalent groups which link thesidechain groups to the peptidic backbone of the compound.

The linking groups L¹ to L⁴ can be groups having a backbone of 1 to 3atoms (e.g., 2 or 3 atoms) that connect adjacent branching groups, e.g.,B¹ and B². A linking group in this context can be an amide bond or amidebond bio isostere. A variety of L¹ to L⁴ can be selected from —CONR′—,—CH₂NR′—, CH(OH)NR′—, —CH₂CONR′—, —CO—, —CH₂CO—, —COCH₂—, —CO₂—, —CH₂O—,—CH₂S—, —CH₂CH₂—, —CH═CH— and —NR′CO—, wherein R′ is H, C₍₁₋₆₎alkyl orsubstituted C₍₁₋₆₎alkyl (e.g., methyl).

It is understood that together the sequence of branching and linkinggroups —B^(n)-L^(n)-represented in the structure of Formula (II) providea peptidic backbone for inhibitors of this disclosure, but that eachindividual —B^(n)-L^(n)- unit in the structure as defined need notnecessarily correspond directly to, or align exactly with, a singlepeptidic unit (A) of a parent peptidic sequence, e.g., sequence (A)_(n)of formula (I). As such, one or more of the —B^(n)(R^(n))-L^(n)- groupsof formula (II), can be selected from the following structures, whereR^(n) refers to a sidechain group, e.g., one of R¹ to R⁴:

The proteasome inhibitors of this disclosure may also conform to one ofthe structures shown in Formulas (IIIa) to (IIIc):

Formula (IIIa) is an exemplary proteasome inhibitor of formula (II) thatincludes peptoid units (e.g., —NR²CH₂CO— monomer units). Formula (IIIa)is an exemplary proteasome inhibitor of formula (II) that includesretroinverso peptidic units, e.g., α-amino acid units configured in areverse sequence as compared to the sequence of α-amino acid units ofFormula (IIIc). Formula (IIIc) is an exemplary proteasome inhibitor offormula (II) that includes α-amino acid residues and has modified N- andC-terminals.

Exemplary is a compound having the structure shown in Formula (Ib):

where:

-   -   X is O or S;    -   R⁰ is selected from H, alkyl, substituted alkyl, alkanoyl,        substituted alkanoyl, alkylaminocarbonyl, substituted        alkylaminocarbonyl, alkoxycarbonyl, substituted alkoxycarbonyl,        alkylaminothiocarbonyl, substituted alkylaminothiocarbonyl,        alkoxythiocarbonyl, substituted alkoxythiocarbonyl and        promoiety;    -   R¹ is selected from alkyl, substituted alkyl, aralkyl,        substituted aralkyl, heteroarylalkyl and substituted        heteroarylalkyl;    -   (AA)_(n) is a sequence of 2 to 7 independently selected amino        acid residues, wherein the C-terminal residue of (AA)_(n)        comprises a modified C-terminal comprising Z; and    -   Z is a proteasome-reactive electrophilic group.

When n is 4 in Formula (Ib), proteasome inhibitors of this disclosuremay also conform to the structure shown in Formula (IIIc)

When used in this context, the term “amino acid” refers either to anaturally occurring or non-naturally occurring amino acid, either in theL- or the D-configuration. For equivalents of the invention, one or moreof the amino acids in the sequence (AA)_(n) may be substituted with anamino acid analog (peptidomimetic unit) linked to the rest of thestructure by way of a covalent bond that is not a peptide bond, whereinthe analog has reactive properties and conformation that aresubstantially the same as the corresponding amino acid.

A “proteasome-reactive electrophilic group” that is part of a proteasomeinhibitor exemplified in this invention is defined as an electrophilicmoiety that upon contact with a target proteasome, reacts with afunctional group (a nucleophilic sidechain on an amino acid residue)near a binding pocket or active site of the proteasome, thereby forminga covalent bond and inhibiting the proteasome from performing itsbiological function. Electrophilic groups that are protein reactiveinclude epoxides, Michael acceptors, disulfides, lactones, b-lactams,and quinones, as taught for example in J. Krysiak and R. Breinbauer, TopCurr Chem (2012) 324: 43-84. See also Chapter 5, pages 207-265 in “Theorganic chemistry of drug design and drug action” by Silverman andHolladay, Third Ed. Academic Press, 2014. For example, reactiveelectrophilic groups that may be used in the proteasome inhibitorsinclude 2-chloro-acetyl (—COCH₂Cl), vinyl sulfone (—SO₂CH═CH₂),acetylene, or methyl-acetylene (i.e., cysteine-reactive groups).

The invention includes compounds according to Formula (I) where Z isselected from epoxyketone group, aziridinylketone group, boronate,boronate ester and beta-lactone. The invention includes compounds ofFormula (I) where (AA)_(n) is a sequence of three independently selectedamino acid residues, wherein the C-terminal residue of (AA)_(n) ismodified to include Z. By modified to include Z can include modifyingthe C-terminal carboxylic acid to a ketone, such as epoxyketone oraziridinylketone, or replacing the C-terminal carboxylic acid with aboronate or boronate ester. Any convenient synthetic methods can beutilized in modifying an amino acid building block to incorporate theproteasome-reactive electrophilic group.

The proteasome inhibitors may also conform to the structure shown inFormula (IV):

where:

-   -   R² to R⁴ are independently selected from alkyl, substituted        alkyl, aralkyl, substituted aralkyl, heteroarylalkyl and        substituted heteroarylalkyl; and    -   Z is epoxide group or aziridine group.

The proteasome inhibitor compounds of Formula (IV) include compounds ofFormula (V):

-   -   where Y is selected from O and NR¹⁵; and R⁵ and R¹⁵ are        independently selected from H, C₍₁₋₆₎alkyl and substituted        C₍₁₋₆₎alkyl.

The invention includes compounds of Formula (II) where R¹ to R⁴ areindependently selected from C₍₁₋₆₎alkyl, substituted C₍₁₋₆₎alkyl,C₍₁₋₆₎hydroxyalkyl, substituted C₍₁₋₆₎hydroxyalkyl,C₍₁₋₆₎alkoxy-C₍₁₋₆₎alkyl, substituted C₍₁₋₆₎alkoxy-C₍₁₋₆₎alkyl,aryl-C₍₁₋₆₎alkyl, substituted aryl-C₍₁₋₆₎alkyl, heteroaryl-C₍₁₋₆₎alkyl,substituted heteroaryl-C₍₁₋₆₎alkyl, cycloalkyl-C₍₁₋₆₎alkyl, substitutedcycloalkyl-C₍₁₋₆₎alkyl, heterocycle-C₍₁₋₆₎alkyl and substitutedheterocycle-C₍₁₋₆₎alkyl.

The proteasome inhibitor compounds can be further described by Formula(VI) or Formula (VII):

In any of the aforelisted structures,

X can be O or S;

R⁰ can be R¹⁰- or R¹⁰-Q-;

Q can be selected from ethylene glycol, polyethylene glycol, —C(═O)—,—NR¹¹C(═O)—, —OC(═O)—, —C(═S)—, —NR¹¹C(═S)—, —OC(═S)— and —OC(═S)—; and

R¹⁰ and R¹ can be independently selected from H, alkyl and substitutedalkyl.

In any of the aforelisted structures, R⁰ may be selected from thefollowing structures:

-   -   where:    -   m is an integer from 1 to 6;    -   p is an integer from 1 to 30;    -   X′ is selected from O, S and NR¹⁴; and    -   R¹² and R¹³ are independently selected from H, alkyl and        substituted alkyl, or R¹² and R¹³ are cyclically linked and        together with the nitrogen atom to which they are attached        provide a heterocycle ring that is optionally further        substituted; and    -   each R¹⁴ is independently selected from H, C₍₁₋₆₎alkyl and        substituted C₍₁₋₆₎alkyl.

In any of the aforelisted structures, R⁰ may be selected from thefollowing structures:

wherein:

-   -   q is an integer from 1 to 3;    -   Y² is selected from O and NR¹⁵; and    -   R¹⁵ is selected from H, C₍₁₋₆₎alkyl and substituted C₍₁₋₆₎alkyl.

In any of the aforelisted structures, R¹ can be selected fromC₍₁₋₆₎alkyl, aryl-C₍₁₋₆₎alkyl, substituted aryl-C₍₁₋₆₎alkyl,cycloalkyl-C₍₁₋₆₎alkyl and substituted cycloalkyl-C₍₁₋₆₎alkyl. R² and R⁴can be selected from C₍₁₋₆₎alkyl, substituted C₍₁₋₆₎alkyl,C₍₁₋₆₎alkoxy-C₍₁₋₆₎alkyl, substituted C₍₁₋₆₎alkoxy-C₍₁₋₆₎alkyl,C₍₁₋₆₎hydroxyalkyl and substituted C₍₁₋₆₎hydroxyalkyl. In addition, R³can be selected from aryl-C₍₁₋₆₎alkyl, substituted aryl-C₍₁₋₆₎alkyl,C₍₁₋₆₎alkoxy-C₍₁₋₆₎alkyl and substituted C₍₁₋₆₎alkoxy-C₍₁₋₆₎alkyl.

In any of the aforelisted structures, R¹ can be selected fromphenyl-C₍₁₋₆₎alkyl, cycloalkyl-C₍₁₋₆₎alkyl and C₍₁₋₆₎alkyl. R² can beselected from C₍₁₋₆₎alkoxy-C₍₁₋₆₎alkyl, C₍₁₋₆₎alkyl andC₍₁₋₆₎hydroxyalkyl. Sometimes, R³ is selected from phenyl-C₍₁₋₆₎alkyl,cycloalkyl-C₍₁₋₆₎alkyl, C₍₁₋₆₎alkoxy-C₍₁₋₆₎alkyl and C₍₁₋₆₎hydroxyalkyl.In addition, R⁴ can be C₍₁₋₆₎alkyl.

In any of the aforelisted structures, R¹ can be selected fromphenylethyl, cyclopropyl-methyl and propyl. R² can be selected frommethoxymethyl, isobutyl and 1-hydroxy-ethyl. Sometimes, R³ is selectedfrom phenylmethyl, cyclopropyl-methyl, methoxymethyl and1-hydroxy-ethyl. In addition, R⁴ can be isobutyl.

In any of the aforelisted structures, R¹ to R⁴ can be selected from oneof combinations #1-6 in the following table:

# R¹ R² R³ R⁴ 1 phenyl-C₍₁₋₆₎alkyl, CH₂CH(CH₃)₂ CH₂Ph CH₂CH(CH₃)₂cycloalkyl-C₍₁₋₆₎alkyl or C₍₁₋₆₎alkyl 2 CH₂Ph C₍₁₋₆₎alkoxy-C₍₁₋₆₎alkyl,CH₂Ph CH₂CH(CH₃)₂ C₍₁₋₆₎alkyl or C₍₁₋₆₎hydroxyalkyl 3 CH₂CH₂PhCH₂CH(CH₃)₂ CH₂CH(CH₃)₂ 4 CH₂CH₂Ph CH₂CH(CH₃)₂ CH₂Ph C₍₁₋₆₎alkyl 5CH₂CH₂Ph C₍₁₋₆₎alkoxy-C₍₁₋₆₎alkyl, CH₂Ph C₍₁₋₆₎alkyl C₍₁₋₆₎alkyl orC₍₁₋₆₎hydroxyalkyl 6 CH₂CH₂Ph CH₂CH(CH₃)₂ CH₂Ph CH₂CH(CH₃)₂

The invention includes proteasome inhibitor compounds according toFormulas (Via) to (VId):

The proteasome inhibitor compounds include compounds according toFormula (VIe) and Formula (VIf). Optionally, Y can be O and R⁵ can bemethyl; X can be O or S.

Examples of proteasome inhibitors are shown in FIGS. 1A, 1B, and 1C. Thetesting and use of selected proteasome inhibitors is provided in thedescription that follows.

Other Proteasome Inhibitors

Besides the peptide-based proteasome inhibitors referred to above, anyproteasome inhibitor currently known in the art or to be developed at alater time can be tested for senolytic activity and developed fortreatment of senescence-associated conditions in accordance with thisdisclosure.

FIG. 1D provides an exemplary list of small molecule compounds that werepreviously described as proteasome inhibitors. Other small moleculeproteasome inhibitors are reviewed in Metcalf et al., Expert Opin TherPat. 2014 April; 24(4):369-8. Any of these compounds are suitable fortesting and development for the purpose of eliminating senescent cellsor treating senescence-associated conditions in accordance with thisdisclosure.

Screening Compounds for Senolytic Activity

The various proteasome inhibitors referred to above and depicted in thedrawings can be screened at the molecular level for their ability toperform in a way that indicate that they are candidate agents for useaccording to this disclosure. Compounds can be tested in molecularassays for their ability to inhibit proteasome activity. Example 1provides assays for this purpose.

Alternatively or in addition, compounds can be screened for an abilityto kill senescent cells specifically. Cultured cells are contacted withthe compound, and the degree of cytotoxicity or inhibition of the cellsis determined. The ability of the compound to kill or inhibit senescentcells can be compared with the effect of the compound on normal cellsthat are freely dividing at low density, and normal cells that are in aquiescent state at high density. Examples 2A and 2B provideillustrations of senescent cell killing using the human target tissuefibroblast IMR90 cell line and HUVEC cells. Similar protocols are knownand can be developed or optimized for testing the ability of the cellsto kill or inhibit other senescent cells and other cell types, such ascancer cells.

FIGS. 2A, 2B, and 2C show results from a screening assay to identifycompounds that selectively kill senescent cells, leaving non-senescentcells intact. FIG. 2A provides data for senolytic activity andproteasome binding for structures selected from FIGS. 1A and 1D. FIG. 2Bprovides data for senolytic activity and proteasome binding forstructures selected from FIG. 1B. FIG. 2C provides data for structuresselected from FIG. 1C.

Candidate senolytic agents that are effective in selectively killingsenescent cells in vitro can be further screened in animal models forparticular disease. Examples 4, 5, 6, and 7 below provide illustrationsfor osteoarthritis, eye disease, lung disease, and atherosclerosis,respectively.

Medicament Formulation and Packaging

Preparation and formulation of pharmaceutical agents for use accordingto this disclosure can incorporate standard technology, as described,for example, in the current edition of Remington: The Science andPractice of Pharmacy. The formulation will typically be optimized foradministration to the target tissue, for example, by localadministration, in a manner that enhances access of the active agent tothe target senolytic cells and providing the optimal duration of effect,while minimizing side effects or exposure to tissues that are notinvolved in the condition being treated.

This invention includes commercial products that are kits that encloseunit doses of one or more of the agents or compositions described inthis disclosure. Such kits typically comprise a pharmaceuticalpreparation in one or more containers. The preparations may be providedas one or more unit doses (either combined or separate). The kit maycontain a device such as a syringe for administration of the agent orcomposition in or around the target tissue of a subject in need thereof.The product may also contain or be accompanied by an informationalpackage insert describing the use and attendant benefits of the drugs intreating the senescent cell associated condition, and optionally anappliance or device for delivery of the composition.

Treatment Design and Dosing Schedule

Senescent cells accumulate with age, which is why conditions mediated bysenescent cells occur more frequently in older adults. In addition,different types of stress on pulmonary tissues may promote the emergenceof senescent cells and the phenotype they express. Cell stressorsinclude oxidative stress, metabolic stress, DNA damage (for example, asa result of environmental ultraviolet light exposure or geneticdisorder), oncogene activation, and telomere shortening (resulting, forexample, from hyperproliferation). Tissues that are subject to suchstressors may have a higher prevalence of senescent cells, which in turnmay lead to presentation of certain conditions at an earlier age, or ina more severe form. An inheritable susceptibility to certain conditionssuggests that the accumulation of disease-mediating senescent cells maydirectly or indirectly be influenced by genetic components, which canlead to earlier presentation.

One of the benefits of the senescent cell paradigm is that successfulremoval of senescent cells may provide the subject with a long-termtherapeutic effect. Senescent cells are essentially non-proliferative,which means that subsequent repopulation of a tissue with more senescentcells can only occur by conversion of non-senescent cells in the tissueto senescent cells—a process that takes considerably longer than simpleproliferation. As a general principle, a period of therapy with asenolytic agent that is sufficient to remove senescent cells from atarget tissue (a single dose, or a plurality of doses given, forexample, every day, semi weekly, or weekly, given over a period of a fewdays, a week, or several months) may provide the subject with a periodof efficacy (for example, for two weeks, a month, two months, or more)during which the senolytic agent is not administered, and the subjectexperiences alleviation, reduction, or reversal of one or more adversesigns or symptoms of the condition being treated.

To treat a particular senescence-related condition with a senolyticagent the therapeutic regimen will depend on the location of thesenescent cells, and the pathophysiology of the disease.

Senescence-Related Conditions Suitable for Treatment

The senolytic agents can be used for prevention or treatment of varioussenescence-related conditions. Such conditions will typically (althoughnot necessarily) characterized by an overabundance of senescent cells(such as cells expressing p16 and other senescence markers) in or aroundthe site of the condition, or an overabundance of expression of p16 andother senescence markers, in comparison with the frequency of such cellsor the level of such expression in unaffected tissue. Non-limitingexamples of current interest include the treatment of osteoarthritis,eye disease, lung disease, and atherosclerosis as illustrated in thefollowing sections.

Treatment of Osteoarthritis

The senolytic agents listed in this disclosure can be developed fortreating osteoarthritis, or for selectively eliminating senescent cellsin or around a joint of a subject in need thereof, including but notlimited to a joint affected by osteoarthritis.

Osteoarthritis degenerative joint disease is characterized byfibrillation of the cartilage at sites of high mechanical stress, bonesclerosis, and thickening of the synovium and the joint capsule.Fibrillation is a local surface disorganization involving splitting ofthe superficial layers of the cartilage. The early splitting istangential with the cartilage surface, following the axes of thepredominant collagen bundles. Collagen within the cartilage becomesdisorganized, and proteoglycans are lost from the cartilage surface. Inthe absence of protective and lubricating effects of proteoglycans in ajoint, collagen fibers become susceptible to degradation, and mechanicaldestruction ensues. Predisposing risk factors for developingosteoarthritis include increasing age, obesity, previous joint injury,overuse of the joint, weak thigh muscles, and genetics. Symptoms ofosteoarthritis include sore or stiff joints, particularly the hips,knees, and lower back, after inactivity or overuse; stiffness afterresting that goes away after movement; and pain that is worse afteractivity or toward the end of the day.

Compounds illustrated in this invention can be used to reduce or inhibitloss or erosion of proteoglycan layers in a joint, reduces inflammationin the affected joint, and promotes, stimulates, enhances, or inducesproduction of collagen, for example, type 2 collagen. The compound maycauses a reduction in the amount, or level, of inflammatory cytokines,such as IL-6, produced in a joint and inflammation is reduced. Thecompounds can be used for treating osteoarthritis and/or inducingcollagen, for example, Type 2 collagen, production in the joint of asubject. A compound also can be used for decreasing, inhibiting, orreducing production of metalloproteinase 13 (MMP-13), which degradescollagen in a joint, and for restoring proteoglycan layer or inhibitingloss and/or degradation of the proteoglycan layer.

Potential benefits of treatment with a senolytic agent includeinhibiting or reversing cartilage or bone erosion. The senolyticcompound may restore or inhibit deterioration of strength of a join, orreduce joint pain.

Treatment of Ophthalmic Conditions

The senolytic agents listed in this disclosure can be used forpreventing or treating an adverse ophthalmic condition in a subject inneed thereof by removing senescent cells in or around an eye of thesubject, whereby at least one sign or symptom of the disease isdecreased in severity. Such conditions include both back-of-the-eyediseases, and front-of-the-eye diseases. The senolytic agents listed inthis disclosure can be developed for selectively eliminating senescentcells in or around ocular tissue in a subject in need thereof.

Diseases of the eye that can be treated include presbyopia, maculardegeneration (including wet or dry AMD), diabetic retinopathy, andglaucoma.

Macular degeneration is a neurodegenerative condition that can becharacterized as a back-of-the-eye disease, It causes the loss ofphotoreceptor cells in the central part of retina, called the macula.Macular degeneration can be dry or wet. The dry form is more common thanthe wet, with about 90% of age-related macular degeneration (AMD)patients diagnosed with the dry form. The wet form of the disease canlead to more serious vision loss. Age and certain genetic factors andenvironmental factors can be risk factors for developing AMD.Environmental factors include, for example, omega-3 fatty acids intake,estrogen exposure, and increased serum levels of vitamin D. Genetic riskfactors can include, for example, reduced ocular Dicer1 levels, anddecreased micro RNAs, and DICER1 ablation.

Dry AMD is associated with atrophy of the retinal pigment epithelium(RPE) layer, which causes loss of photoreceptor cells. The dry form ofAMD can result from aging and thinning of macular tissues and fromdeposition of pigment in the macula. With wet AMD, new blood vessels cangrow beneath the retina and leak blood and fluid. Abnormally leakychoroidal neovascularization can cause the retinal cells to die,creating blind spots in central vision. Different forms of maculardegeneration can also occur in younger patients. Non-age relatedetiology can be linked to, for example, heredity, diabetes, nutritionaldeficits, head injury, or infection.

The formation of exudates, or “drusen,” underneath the Bruch's membraneof the macula is can be a physical sign that macular degeneration candevelop. Symptoms of macular degeneration include, for example,perceived distortion of straight lines and, in some cases, the center ofvision appears more distorted than the rest of a scene; a dark, blurryarea or “white-out” appears in the center of vision; or color perceptionchanges or diminishes.

Another back-of-the-eye disease is diabetic retinopathy (DR). Accordingto Wikipedia, the first stage of DR is non-proliferative, and typicallyhas no substantial symptoms or signs. NPDR is detectable by fundusphotography, in which microaneurysms (microscopic blood-filled bulges inthe artery walls) can be seen. If there is reduced vision, fluoresceinangiography can be done to see the back of the eye. Narrowing or blockedretinal blood vessels can be seen clearly and this is called retinalischemia (lack of blood flow). Macular edema in which blood vessels leaktheir contents into the macular region can occur at any stage of NPDR.The symptoms of macular edema are blurred vision and darkened ordistorted images that are not the same in both eyes. Ten percent (10%)of diabetic patients will have vision loss related to macular edema.Optical Coherence Tomography can show the areas of retinal thickening(due to fluid accumulation) of macular edema.

In the second stage of DR, abnormal new blood vessels(neovascularization) form at the back of the eye as part ofproliferative diabetic retinopathy (PDR); these can burst and bleed(vitreous hemorrhage) and blur the vision, because these new bloodvessels are fragile. The first time this bleeding occurs, it may not bevery severe. In most cases, it will leave just a few specks of blood, orspots floating in a person's visual field, though the spots often goaway after few hours. These spots are often followed within a few daysor weeks by a much greater leakage of blood, which blurs the vision. Inextreme cases, a person may only be able to tell light from dark in thateye. It may take the blood anywhere from a few days to months or evenyears to clear from the inside of the eye, and in some cases the bloodwill not clear. These types of large hemorrhages tend to happen morethan once, often during sleep. On funduscopic exam, a doctor will seecotton wool spots, flame hemorrhages (similar lesions are also caused bythe alpha-toxin of Clostridium novyi), and dot-blot hemorrhages.

Presbyopia is an age-related condition where the eye exhibits aprogressively diminished ability to focus on near objects as the speedand amplitude of accommodation of a normal eye decreases with advancingage. Loss of elasticity of the crystalline lens and loss ofcontractility of the ciliary muscles can cause presbyopia. Age-relatedchanges in the mechanical properties of the anterior lens capsule andposterior lens capsule suggest that the mechanical strength of theposterior lens capsule decreases significantly with age. The laminatedstructure of the capsule of the eye also changes and can result, atleast in part, from a change in the composition of the tissue.

Compounds provided by this disclosure can slow the disorganization ofthe type IV collagen network, decrease or inhibit epithelial cellmigration and can also delay the onset of presbyopia or decrease or slowthe progressive severity of the condition. They can also be useful forpost-cataract surgery to reduce the likelihood of occurrence of PCO.

Another condition treatable with senolytic agents is glaucoma. Normally,clear fluid flows into and out of the front part of the eye, known asthe anterior chamber. In individuals who have open/wide-angle glaucoma,the clear fluid drains too slowly, leading to increased pressure withinthe eye. If left untreated, the high pressure in the eye cansubsequently damage the optic nerve and can lead to complete blindness.The loss of peripheral vision is caused by the death of ganglion cellsin the retina. The effect of a therapy on inhibiting progression ofglaucoma can be monitored by automated perimetry, gonioscopy, imagingtechnology, scanning laser tomography, HRT3, laser polarimetry, GDX,ocular coherence tomography, ophthalmoscopy, and pachymeter measurementsthat determine central corneal thickness.

Ophthalmic conditions treatable with senolytic agents include ischemicor vascular conditions, such as diabetic retinopathy, glaucomatousretinopathy, ischemic arteritic optic neuropathies, and vasculardiseases characterized by arterial and venous occlusion, retinopathy ofprematurity and sickle cell retinopathy.

Ophthalmic conditions treatable with senolytic agents includedegenerative conditions, such as dermatochalasis, ptosis, keratitissicca, Fuch's corneal dystrophy, presbyopia, cataract, wet age relatedmacular degeneration (wet AMD), dry age related macular degeneration(dry AMD); degenerative vitreous disorders, including vitreomaculartraction (VMT) syndrome, macular hole, epiretinal membrane (ERM),retinal tears, retinal detachment, and proliferative vitreoretinopathy(PVR).

Ophthalmic conditions treatable with senolytic agents include geneticconditions, such as retinitis pigmentosa, Stargardt disease, Bestdisease and Leber's hereditary optic neuropathy (LHON). Ophthalmicconditions treatable with a senolytic agent include conditions caused bya bacterial, fungal, or virus infection. These include conditions causedor provoked by an etiologic agent such as herpes zoster varicella (HZV),herpes simplex, cytomegalovirus (CMV), and human immunodeficiency virus(HIV).

Ophthalmic conditions treatable with senolytic agents includeinflammatory conditions, such as punctate choroiditis (PIC), multifocalchoroiditis (MIC) and serpiginous choroidopathy. Ophthalmic conditionstreatable with a senolytic agent also include iatrogenic conditions,such as a post-vitrectomy cataract and radiation retinopathy.

Potential benefits of treatment with a senolytic agent include reversingor inhibiting progression of any of the aforelisted signs and symptomsof ocular diseases, such as neovascularization, vaso-obliteration, andan increase in intraocular pressure, leading to an impairment of retinalfunction and loss of vision.

Treatment of Pulmonary Conditions

The senolytic agents listed in this disclosure can be developed fortreating pulmonary disease, or for selectively eliminating senescentcells in or around a lung of a subject in need thereof. Pulmonaryconditions that can be treated include idiopathic pulmonary fibrosis(IPF), chronic obstructive pulmonary disease (COPD), asthma, cysticfibrosis, bronchiectasis, and emphysema.

COPD is a lung disease defined by persistently poor airflow resultingfrom the breakdown of lung tissue, emphysema, and the dysfunction of thesmall airways, obstructive bronchiolitis. Primary symptoms of COPDinclude shortness of breath, wheezing, chest tightness, chronic cough,and excess sputum production. Elastase from cigarette smoke-activatedneutrophils and macrophages can disintegrate the extracellular matrix ofalveolar structures, resulting in enlarged air spaces and loss ofrespiratory capacity. COPD can be caused by, for example, tobacco smoke,cigarette smoke, cigar smoke, secondhand smoke, pipe smoke, occupationalexposure, exposure to dust, smoke, fumes, and pollution, occurring overdecades thereby implicating aging as a risk factor for developing COPD.High concentrations of free radicals in tobacco smoke can lead tocytokine release as part of an inflammatory response to irritants in theairway, resulting in damage the lungs by protease.

Symptoms of COPD can include shortness of breath, wheezing, chesttightness, having to clear one's throat first thing in the morningbecause of excess mucus in the lungs, a chronic cough that producessputum that can be clear, white, yellow or greenish, cyanosis, frequentrespiratory infections, lack of energy, and unintended weight loss.

Pulmonary fibrosis is a chronic and progressive lung diseasecharacterized by stiffening and scarring of the lung, which can lead torespiratory failure, lung cancer, and heart failure. Fibrosis isassociated with repair of epithelium. Fibroblasts are activated,production of extracellular matrix proteins is increased, andtransdifferentiation to contractile myofibroblasts contribute to woundcontraction. A provisional matrix plugs the injured epithelium andprovides a scaffold for epithelial cell migration, involving anepithelial-mesenchymal transition (EMT). Blood loss associated withepithelial injury induces platelet activation, production of growthfactors, and an acute inflammatory response. Normally, the epithelialbarrier heals and the inflammatory response resolves. However, infibrotic disease the fibroblast response continues, resulting inunresolved wound healing. Formation of fibroblastic foci is a feature ofthe disease, reflecting locations of ongoing fibrogenesis.

Subjects at risk of developing pulmonary fibrosis include, for example,those exposed to environmental or occupational pollutants, such asasbestosis and silicosis; those who smoke cigarettes; those who have aconnective tissue diseases such as RA, SLE, scleroderma, sarcoidosis, orWegener's granulomatosis; those who have infections; those who takecertain medications, including, for example, amiodarone, bleomycin,busufan, methotrexate, and nitrofurantoin; those subject to radiationtherapy to the chest; and those whose family member have pulmonaryfibrosis.

Other pulmonary conditions that can be treated by using a compoundinclude emphysema, asthma, bronchiectasis, and cystic fibrosis.Pulmonary diseases can also be exacerbated by tobacco smoke,occupational exposure to dust, smoke, or fumes, infection, or pollutantsthat contribute to inflammation.

Bronchiectasis can result from damage to the airways that causes them towiden and become flabby and scarred. Bronchiectasis can be caused by amedical condition that injures the airway walls or inhibits the airwaysfrom clearing mucus. Examples of such conditions include cystic fibrosisand primary ciliary dyskinesia (PCD). When only one part of the lung isaffected, the disorder can be caused by a blockage rather than a medicalcondition.

The methods provided in this disclosure for treating or reducing thelikelihood of a pulmonary condition can also be used for treating asubject who is aging and has loss of pulmonary function, or degenerationof pulmonary tissue. Effects of treatment can be determined usingtechniques that evaluate mechanical functioning of the lung, forexample, techniques that measure lung capacitance, elastance, and airwayhypersensitivity can be performed. For example, expiratory reservevolume (ERV), forced vital capacity (FVC), forced expiratory volume(FEV) (e.g., FEV in one second, FEV1), FEV1/FEV ratio, forced expiratoryflow 25% to 75%, and maximum voluntary ventilation (MVV), peakexpiratory flow (PEF), slow vital capacity (SVC) can be measured.Peripheral capillary oxygen saturation (SpO₂) can also be measured;normal oxygen levels are typically between 95% and 100%. An SpO₂ levelbelow 90% indicates that the subject has hypoxemia.

Potential benefits of treatment with a senolytic agent include includealleviating or halting progression of one or more signs or symptoms ofthe condition being treated, as indicated above. Objectives may includeincreasing lung volume or capacity, and manifestations thereof such asimproving oxygen saturation.

Treatment of Atherosclerosis

The senolytic compounds can be used for the treatment ofatherosclerosis: for example, by inhibiting formation, enlargement, orprogression of atherosclerotic plaques in a subject. The senolyticcompounds can also be used to enhance stability of atheroscleroticplaques that are present in one or more blood vessels of a subject,thereby inhibiting them from rupturing and occluding the vessels.

Atherosclerosis is characterized by patchy intimal plaques, atheromas,that encroach on the lumen of medium-sized and large arteries; theplaques contain lipids, inflammatory cells, smooth muscle cells, andconnective tissue. Atherosclerosis can affect large and medium-sizedarteries, including the coronary, carotid, and cerebral arteries, theaorta and branches thereof, and major arteries of the extremities.

Atherosclerosis may lead to an increase in artery wall thickens.Symptoms develop when growth or rupture of the plaque reduces orobstructs blood flow; and the symptoms can vary depending on whichartery is affected. Atherosclerotic plaques can be stable or unstable.Stable plaques regress, remain static, or grow slowly, sometimes overseveral decades, until they can cause stenosis or occlusion. Unstableplaques are vulnerable to spontaneous erosion, fissure, or rupture,causing acute thrombosis, occlusion, and infarction long before theycause hemodynamically significant stenosis. Clinical events can resultfrom unstable plaques, which do not appear severe on angiography; thus,plaque stabilization can be a way to reduce morbidity and mortality.Plaque rupture or erosion can lead to major cardiovascular events suchas acute coronary syndrome and stroke. Disrupted plaques can have agreater content of lipid, macrophages, and have a thinner fibrous capthan intact plaques.

Atherosclerosis is thought to be due in significant part to a chronicinflammatory response of white blood cells in the walls of arteries.This is promoted by low-density lipoproteins (LDL), plasma proteins thatcarry cholesterol and triglycerides, in the absence of adequate removalof fats and cholesterol from macrophages by functional high-densitylipoproteins (HDL). The earliest visible lesion of atherosclerosis isthe “fatty streak,” which is an accumulation of lipid-laden foam cellsin the intimal layer of the artery. The hallmark of atherosclerosis isatherosclerotic

Diagnosis of atherosclerosis and other cardiovascular disease can bebased on symptoms, for example, angina, chest pressure, numbness orweakness in arms or legs, difficulty speaking or slurred speech,drooping muscles in face, leg pain, high blood pressure, kidney failureand/or erectile dysfunction, medical history, and/or physicalexamination of a patient. Diagnosis can be confirmed by angiography,ultrasonography, or other imaging tests. Subjects at risk of developingcardiovascular disease include those having any one or more ofpredisposing factors, such as a family history of cardiovascular diseaseand those having other risk factors, for example, predisposing factorsincluding high blood pressure, dyslipidemia, high cholesterol, diabetes,obesity and cigarette smoking, sedentary lifestyle, and hypertension.The condition can be assessed, for example, by angiography,electrocardiography, or stress test.

Potential benefits of treatment with a senolytic agent includealleviating or halting progression of one or more signs or symptoms ofthe condition, such as the frequency of plaques, the surface area ofvessels covered by plaques, angina, and reduced exercise tolerance.

Definitions

A “senescent cell” is generally thought to be derived from a cell typethat typically replicates, but as a result of aging or other event thatcauses a change in cell state, can no longer replicate. For the purposeof practicing some aspects of this invention, senescent cells can beidentified as expressing p16, or at least one marker selected from p16,senescence-associated β-galactosidase, and lipofuscin; sometimes two ormore of these markers, and other markers of the senescence-associatedsecretory profile (SASP) such as but not limited to interleukin 6, andinflammatory, angiogenic and extracellular matrix modifying proteins.Unless explicity stated otherwise, the senescent cells referred to inthe claims do not include cancer cells.

A “senescence associated”, “senescence related” or “age related”disease, disorder, or condition is a physiological condition thatpresents with one or more symptoms or signs that are adverse to thesubject. The condition is “senescence associated” if it is “caused ormediated at least in part by senescent cells.” This means that at leastone component of the SASP in or around the affected tissue plays a rolein the pathophysiology of the condition such that elimination of atleast some of the senescent cells in the affected tissue results insubstantial relief or lessening of the adverse symptoms or signs, to thepatient's benefit. Senescence associated disorders that can potentiallybe treated or managed using the methods and products according to thisinvention include disorders referred to in this disclosure and inprevious disclosures referred to in the discussion. Unless explicitlystated otherwise, the term does not include cancer.

An inhibitor of protein function or proteasome function is a compoundthat to a substantial degree prevents the target protein alreadyexpressed in a target cell from performing an enzymatic, binding, orregulatory function that the protein or proteasome normally performs inthe target cell. This results in elimination of the target cell orrendering the cell more susceptible to the toxicity of another compoundor event. A compound qualifies as a “proteasome inhibitor” or a compoundthat “inhibits proteasome activity” in this disclosure if it has an IC₅₀when tested in an assay according to Example 1 below (exemplified inFIGS. 2A, 2B, and 2C) that is less than 1,000 nM (1.0 μM). Activity thatis less than 100 nM or 10 nM, or between 100 nM and 1 nM is oftenpreferred, depending on the context.

A compound, composition or agent is typically referred to as “senolytic”if it eliminates senescent cells, compared with replicative cells of thesame tissue type, or quiescent cells lacking SASP markers. Alternativelyor in addition, a compound or combination may effectively be used if itdecreases the release of pathological soluble factors or mediators aspart of the senescence associated secretory phenotype that play a rolein the initial presentation or ongoing pathology of a condition, orinhibit its resolution. In this respect, the term “senolytic” refers tofunctional inhibition, such that compounds that work primarily byinhibiting rather than eliminating senescent cells (senescent cellinhibitors) can be used in a similar fashion with ensuing benefits.Model senolytic compositions and agents in this disclosure have an EC₅₀when tested in an assay according to Example 2 below (exemplified inFIGS. 2A, 2B, and 2C) that is less than 1 μM. Activity that is less than0.1 μM, or between 1 μM and 0.1 μM may be preferred. The selectivityindex (SI) (EC₅₀ of senescent cells compared with non-senescent cells ofthe same tissue type) may be 1, 2, 5, or 10 or more, depending on thecontext.

Selective removal or “elimination” of senescent cells from a mixed cellpopulation or tissue doesn't require that all cells bearing a senescencephenotype be removed: only that the proportion of senescent cellsinitially in the tissue that remain after treatment is substantiallyhigher than the proportion of non-senescent cells initially in thetissue that remain after the treatment.

Successful “treatment” of a condition may have any effect that isbeneficial to the subject being treated. This includes decreasingseverity, duration, or progression of a condition, or of any adversesigns or symptoms resulting therefrom. In some circumstances, senolyticagents can also be used to prevent or inhibit presentation of acondition for which a subject is susceptible, for example, because of aninherited susceptibility of because of medical history.

A “therapeutically effective amount” is an amount of a compound of thepresent disclosure that (i) treats the particular disease, condition, ordisorder, (ii) attenuates, ameliorates, or eliminates one or moresymptoms of the particular disease, condition, or disorder, (iii)prevents or delays the onset of one or more symptoms of the particulardisease, condition, or disorder described herein, (iv) prevents ordelays progression of the particular disease, condition or disorder, (v)at least partially reverses damage caused by the condition prior totreatment; or has a plurality of such effects in any combination.

A “phosphorylated” form of a compound is a compound which bears one ormore phosphate groups covalently bound to the core structure through anoxygen atom, which was typically but not necessarily present on themolecule before phosphorylation. For example, one or more —OH or —COOHgroups may have been substituted in place of the hydrogen with aphosphate group which is either —OPO₃H₂ or —C_(n)PO₃H₂ (where n is 1 to4). In some phosphorylated forms, the phosphate group may be removed invivo (for example, by enzymolysis), in which case the phosphorylatedform may be a pro-drug of the non-phosphorylated form. Anon-phosphorylated form has no such phosphate group. A dephosphorylatedform is a derivative of a phosphorylated molecule after at least onephosphate group has been removed.

“Peptidic” compounds of this invention refers collectively or in thealternative to compounds that contains either a normal peptide withamino acids linked together by peptide bonds, or that contain apeptidomimetic portion that is capable of mimicking a biological actionof a parent peptide. A “peptidomimetic” compound is a bioisostere of aparent peptide sequence that contains structural elements that arepositioned in three-dimensional space to have chemical effects atlocations that mimic the chemical effects of corresponding structuralelements of the parent peptide. As a result, the peptidomimetic compoundbinds to or interacts with a target biological molecule in a way thatmimics an activity of the parent peptide, while typically also having adesirable physical and/or non-target biological property that differsfrom the parent peptide, such as resistance to proteolytic degradationor increased bioavailability. A peptidomimetic compound typicallyincludes a backbone and a configuration of side chains corresponding tothe peptide backbone and side chains of the parent peptide. Included areretrooinverso peptides, peptoids, and other backbones that present sidechains in similar fashion to a parent peptide, such as substitutions ofthe amide bond hydrogen moiety by methyl groups (N-methylation) or otheralkyl groups. A peptidic compound can have a portion that is a normalpolypeptide, and a portion that is a peptidomimetic sequence or monomer.A peptidic compound may have other reactive groups or modifications ateither terminal of the backbone or on one or more side chains. Possiblemodifications include an N-terminal modification (such as an oxo or thiogroup) and a C-terminal modification (such as a C-terminal epoxyketone).

A “retroinverso” peptide is a sequence of D-amino acid residues that isthe same as the sequence of a parent L-peptide, except it is configuredin the reverse order. A retroinverso peptide can maintain an amino acidsidechain topology that is similar to that of the parent L-amino acidpeptide and be more resistant to proteolytic degradation. A “peptoid” isa class of peptidomimetic compounds, or a unit thereof, based onN-substituted glycine monomer units where the sidechain groups arelinked to the nitrogen atom of the peptidic backbone. Peptoid compoundscan be designed to display sidechain groups analogous to the bioactivepeptide side chains of a parent peptide sequence, while the peptoidbackbone can provide resistance to proteolytic degradation.

“Small molecule” senolytic agents have molecular weights less than20,000 daltons, and are often less than 10,000, 5,000, or 2,000 daltons.Small molecule inhibitors are not antibody molecules oroligonucleotides, and typically have no more than five hydrogen bonddonors (the total number of nitrogen-hydrogen and oxygen-hydrogenbonds), and no more than 10 hydrogen bond acceptors that are nitrogen oroxygen atoms.

“Prodrug” refers to a derivative of an active agent that requires atransformation within the body to release the active agent. Thetransformation can be an enzymatic transformation. Sometimes, thetransformation is a cyclization transformation, or a combination of anenzymatic transformation and a cyclization transformation. Prodrugs arefrequently, although not necessarily, pharmacologically inactive untilconverted to the active agent.

“Promoiety” refers to a form of protecting group that when used to maska functional group within an active agent converts the active agent intoa prodrug. Typically, the promoiety will be attached to the drug viabond(s) that are cleaved by enzymatic or non-enzymatic means in vivo.Exemplary promoiety groups include acyl groups capable of forming anester or thioester group with a hydroxyl or thiol functional group of acompound, and substituted alkyl groups capable of forming an ether orthioether group with a hydroxyl or thiol functional group of a compound,which groups can be cleaved in vivo as described above.

Unless otherwise stated or required, each of the compound structuresreferred to in the invention include conjugate acids and bases havingthe same structure, crystalline and amorphous forms of those compounds,pharmaceutically acceptable salts, and prodrugs. This includes, forexample, tautomers, polymorphs, solvates, hydrates, unsolvatedpolymorphs (including anhydrates). The compound may be any stereoisomerof the structure shown, or a mixture thereof, unless a particularstereoisomer or a particular chiral structure is explicity referred to.

Unless otherwise stated or implied, the term “substituted” when used tomodify a specified group or radical means that one or more hydrogenatoms of the specified group or radical are each independently replacedwith the same or different substituent groups which is not hydrogen.Unless indicated otherwise, the nomenclature of substituents is arrivedat by naming the terminal portion of the functionality followed by theadjacent functionality toward the point of attachment. For example, thesubstituent “arylalkyloxycarbonyl” refers to the group(aryl)-(alkyl)-O—C(O)—.

A “linker” is a moiety that covalently connects two or more chemicalstructures, and has a backbone of 100 atoms or less in length betweenthe two structures. The linker may be cleavable or non-cleavable. Thelinker typically has a backbone of between 1 and 20 or between 1 and 100atoms in length, in linear or branched form. The bonds between backboneatoms may be saturated or unsaturated. The linker backbone may include acyclic group, for example, an optionally substituted aryl, heteroaryl,heterocycle or cycloalkyl group.

Except where otherwise stated or required, other terms used in thespecification have their ordinary meaning.

INCORPORATION BY REFERENCE

For all purposes in the United States and in other jurisdictions whereeffective, each and every publication and patent document cited in thisdisclosure is hereby incorporated herein by reference in its entiretyfor all purposes to the same extent as if each such publication ordocument was specifically and individually indicated to be incorporatedherein by reference.

US 2016/0339019 A1 (Laberge et al.) and US 20170266211 A1 (David et al.)are hereby incorporated herein by reference in their entirety for allpurposes, including but not limited to the identification, formulation,and use of compounds for eliminating or reducing the activity ofsenescent cells and treating particular senescence-related conditions,including but not limited to those referred to in this disclosure. U.S.patent applications US 2018/0000816 A1 and PCT/US2018/046553 are herebyincorporated herein for all purposes, including but not limited to theidentification, formulation, and use of compounds for eliminating orreducing the activity of senescent cells and treating various ophthalmicconditions. U.S. patent applications US 2018/0000816 A1 andPCT/US2018/046567 are hereby incorporated herein for all purposes,including but not limited to the identification, formulation, and use ofcompounds for eliminating or reducing the activity of senescent cellsand treating various pulmonary conditions. U.S. patent application Ser.No. 16/181,163 are hereby incorporated herein for all purposes,including but not limited to the identification, formulation, and use ofcompounds for eliminating or reducing the activity of senescent cellsand treating atherosclerosis.

EXAMPLES Example 1: Measuring Proteasome Activity

This example provides assays by which the reader may ascertain whether atest compound has sufficient inhibitory capacity for the target pathwaysto be developed as a senolytic agent. Information from these assays maybe combined with information from cell lysis assays (Examples 2 and 3)to select compounds for further development.

Test compounds are assayed for inhibition of the chymotrypsin-likeactivity of the proteasome 35 subunit by monitoring the release of afluorogenic product after cleavage of a substrate peptide. The activeprotease cleaves an amide bond between the C-terminal amino acid of asubstrate peptide and aminoemethylcoumarin, allowing enzyme activity tobe quantitated fluorometrically.

Compounds are tested in a 384-well format. A 1:3 dilution series ofcompound in DMSO is diluted into reaction buffer (20 mM HEPES pH 7.5,0.01% BSA, 0.02% SDS, 0.5 mM EDTA, 100 mM NaCl) so that when added tothe reaction mix the final concentration of DMSO does not exceed 1%. Toinitialize the reaction, test compounds and substrate are added for afinal reaction volume of 50 μL per well containing the following: 20 mMHEPES pH 7.5, 0.01% BSA, 0.02% SDS, 0.5 mM EDTA, 100 mM NaCl, 0.5 nMconstitutive 20S proteasome, and 50 μM substrate(Succinyl-Leu-Leu-Val-Tyr-AMC).

Reactions are mixed and an initial reading is recorded after 5 minutesusing and excitation wavelength of 360 nM and emission of 450 nM. Asecond endpoint measurement is taken at 1 hour. Relative enzymaticactivity is calculated from the change in fluorescence (final minusinitial) relative to DMSO control.

Example 2A: Measuring Senolytic Activity in Fibroblasts

Before initiating experiments in vivo, it is usually helpful to screenpotential senolytic agents for their potency for removing senescentcells, and their selectivity for senescent cells in comparison withnon-senescent cells in the same tissue.

Human fibroblast IMR90 cells can be obtained from the American TypeCulture Collection (ATCC®) with the designation CCL-186. The cells aremaintained at <75% confluency in DMEM containing FBS and Pen/Strep in anatmosphere of 3% O2, 10% CO2, and ˜95% humidity. The cells are dividedinto three groups: irradiated cells (cultured for 14 days afterirradiation prior to use), proliferating normal cells (cultured at lowdensity for one day prior to use), and quiescent cells (cultured at highdensity for four day prior to use).

On day 0, the irradiated cells are prepared as follows. IMR90 cells arewashed, placed in T175 flasks at a density of 50,000 cells per mL, andirradiated at 10-15 Gy. Following irradiation, the cells are plated at100 μL in 96-well plates. On days 1, 3, 6, 10, and 13, the medium ineach well is aspirated and replaced with fresh medium.

On day 10, the quiescent healthy cells are prepared as follows. IMR90cells are washed, combined with 3 mL of TrypLE trypsin-containingreagent (Thermofisher Scientific, Waltham, Mass.) and cultured for 5 minuntil the cells have rounded up and begin to detach from the plate.Cells are dispersed, counted, and prepared in medium at a concentrationof 50,000 cells per mL. 100 μL of the cells is plated in each well of a96-well plate. Medium is changed on day 13.

On day 13, the proliferating healthy cell population is prepared asfollows. Healthy IMR90 cells are washed, combined with 3 mL of TrypLEand cultured for 5 minutes until the cells have rounded up and begin todetach from the plate. Cells are dispersed, counted, and prepared inmedium at a concentration of 25,000 cells per mL. 100 μL of the cells isplated in each well of a 96-well plate.

On day 14, test inhibitors are combined with the cells as follows. ADMSO dilution series of each test compound is prepared at 200 times thefinal desired concentration in a 96-well PCR plate. Immediately beforeuse, the DMSO stocks are diluted 1:200 into pre-warmed complete medium.Medium is aspirated from the cells in each well, and 100 μL/well of thecompound containing medium is added.

Candidate senolytic agents for testing are cultured with the cells for 6days, replacing the culture medium with fresh medium and the samecompound concentration on day 17. Test inhibitors are cultured with thecells for 3 days. The assay system uses the properties of a thermostableluciferase to enable reaction conditions that generate a stableluminescent signal while simultaneously inhibiting endogenous ATPasereleased during cell lysis. At the end of the culture period, 100 μL ofCellTiter-Glo® reagent (Promega Corp., Madison, Wis.) is added to eachof the wells. The cell plates are placed for 30 seconds on an orbitalshaker, and luminescence is measured.

FIG. 2A provides data for senolytic activity and proteasome binding forstructures selected from FIGS. 1A and 1D. FIG. 2B provides data forsenolytic activity and proteasome binding for structures selected fromFIG. 1B. FIG. 2C provides data for structures selected from FIG. 1C

Example 2B: Measuring Senolytic Activity in HUVEC Cells

Human umbilical vein (HUVEC) cells from a single lot were expanded inVascular Cell Basal Media supplemented with the Endothelial Cell GrowthKit™-VEGF from ATCC to approximately eight population doublings thencryopreserved. Nine days prior to the start of the assay, cells for thesenescent population were thawed and seeded at approximately 27,000/cm2.All cells were cultured in humidified incubators with 5% CO2 and 3% 02and media was changed every 48 hr. Two days after seeding, the cellswere irradiated, delivering 12 Gy radiation from an X-ray source. Threedays prior to the start of the assay, cells for the non-senescentpopulations are thawed and seeded as for the senescent population. Oneday prior to the assay, all cells were trypsinized and seeded into384-well plates, 5,000/well senescent cells and 10,000/wellnon-senescent in separate plates in a final volume of 55 μL/well. Ineach plate, the central 308 wells contained cells and the outerperimeter of wells was filled with 70 μL/well deionized water.

On the day of the assay, compounds were diluted from 10 mM stocks intomedia to provide the highest concentration working stock, aliquots ofwhich were then further diluted in media to provide the remaining twoworking stocks. To initiate the assay, 5 μL of the working stock wasadded to the cell plates. The final test concentrations were 20, 2, and0.2 μM. In each plate, 100 test compounds were assayed in triplicate ata single concentration along with a three wells of a positive controland five no treatment (DMSO) controls. Following compound addition, theplates are returned to the incubators for three days.

Cell survival was assessed indirectly by measuring total ATPconcentration using CellTiter-Glo™ reagent (Promega). The resultantluminescence was quantitated with an EnSpire™ plate reader (PerkinElmer). The relative cell viability for each concentration of a compoundwas calculated as a percentage relative to the no-treatment controls forthe same plate.

For follow-up dose responses of potential lead compounds, 384-wellplates of senescent and non-senescent cells were prepared as describedabove. Compounds were prepared as 10-point 1:3 dilution series in DMSO,then diluted to 12× in media. Five microliters of this working stock wasthen added to the cell plates. After three days of incubation, cellsurvival relative to DMSO control was calculated as described above. Allmeasurements were preformed in quadruplicate.

Example 3: Synthesis of Proteasome Inhibitors

General Synthetic Scheme:

This scheme can be applied to the synthesis of(S)-2-((S)-2-hydroxy-4-phenylbutanamido)-4-methyl-N—((S)-1-(((S)-4-methyl-1-((R)-2-methyloxiran-2-yl)-1-oxopentan-2-yl)amino)-1-oxo-3-phenylpropan-2-yl)pentanamide

Step 1: Synthesis of benzyl(tert-butoxycarbonyl)-L-leucyl-L-phenylalaninate

The mixture of (tert-butoxycarbonyl)-L-leucine (10.2 g, 41.0 mmol),benzyl L-phenylalaninate (10.0 g, 34.2 mmol) and TEA (8.63 g, 85.5 mmol)in DCM (150 mL) was added T₃P (32.6 g, 51.3 mmol, 50% of EtOAcsolution), then the mixture was stirred at 25° C. for 1 h. The reactionmixture was washed with water (200 mL×3), dried over Na₂SO₄,concentrated in vacuum. The residue was purified by column (EA in PEfrom 0% to 25%) to afford benzyl(tert-butoxycarbonyl)-L-leucyl-L-phenylalaninate (10.0 g, yield: 48.4%)as white solid.

Step 2: Synthesis of benzyl L-leucyl-L-phenylalaninate

To a solution of benzyl (tert-butoxycarbonyl)-L-leucyl-L-phenylalaninate(30.0 g, 64.0 mmol) in DCM (500 mL) was added TFA (100 mL) at 0° C.After 4 h at room temperature, the solvent was removed under vacuum.MTBE (200 mL) was added and the solid was filtered and washed with MTBE(100 mL×3), then dried in vacuo to afford benzylL-leucyl-L-phenylalaninate (30.0 g, 62.1 mmol) as a white solid; LCMS(ESI⁺) [(M+H)⁺]: 369.1

Step 3: Synthesis of benzyl((S)-2-hydroxy-4-phenylbutanoyl)-L-leucyl-L-phenylalaninate

A mixture of benzyl L-leucyl-L-phenylalaninate (8.00 g, 16.5 mmol),(S)-2-hydroxy-4-phenylbutanoic acid (3.40 g, 18.9 mmol), DIPEA (4.69 g,36.3 mmol) and BOP (10.9 g, 24.7 mmol) in DCM (150 mL) was stirred at25° C. for 16 h. The reaction mixture was concentrated under vacuum.Preparative HPLC afforded benzyl((S)-2-hydroxy-4-phenylbutanoyl)-L-leucyl-L-phenylalaninate (14.0 g,yield: 60.8%) was obtained as white solid; LCMS (ESI⁺) [(M+H)⁺]: 531.5

Step 4: Synthesis of((S)-2-hydroxy-4-phenylbutanoyl)-L-leucyl-L-phenylalanine

A mixture of benzyl((S)-2-hydroxy-4-phenylbutanoyl)-L-leucyl-L-phenylalaninate (12.0 g,22.6 mmol) and Pd/C (2 g, 10%) in EtOAc (400 mL) was stirred under H₂(15 psi) at 25° C. for 4 h. The reaction mixture was filtered andconcentrated in vacuo to afford((S)-2-hydroxy-4-phenylbutanoyl)-L-leucyl-L-phenylalanine (10 g, yield:99.0%) as white solid; LCMS (ESI⁺) [(M+H)⁺]: 441.4

Step 5: Synthesis of(S)-2-((S)-2-hydroxy-4-phenylbutanamido)-4-methyl-N—((S)-1-(((S)-4-methyl-1-((R)-2-methyloxiran-2-yl)-1-oxopentan-2-yl)amino)-1-oxo-3-phenylpropan-2-yl)pentanamide

T₃P (10.7 g, 16.9 mmol) was slowly added to a solution of((S)-2-hydroxy-4-phenylbutanoyl)-L-leucyl-L-phenylalanine (5.00 g, 11.3mmol), (S)-2-amino-4-methyl-1-((R)-2-methyloxiran-2-yl)pentan-1-one(3.53 g, 12.4 mmol) and TEA (2.50 g, 24.8 mmol) in MeCN (100 mL). After1 h, H₂O (80 mL) was added to the suspension and stirred for 5 min. Themixture was filtered and the solid was washed with H₂O/MeCN (50 mL,1:1), then dried under high vacuum to afford(S)-2-((S)-2-hydroxy-4-phenylbutanamido)-4-methyl-N—((S)-1-(((S)-4-methyl-1-((R)-2-methyloxiran-2-yl)-1-oxopentan-2-yl)amino)-1-oxo-3-phenylpropan-2-yl)pentanamide(5.1 g); LCMS (ESI⁺) [(M+H)⁺]: 594.7

Example 4: Efficacy of Senolytic Agents in an Osteoarthritis Model

This example illustrates the testing of an MDM2 inhibitor in a mousemodel for treatment of osteoarthritis. It can be adapted mutatismutandis to test and develop senolytic agents for use in clinicaltherapy.

The model was implemented as follows. C57BL/6J mice underwent surgery tocut the anterior cruciate ligament of one rear limb to induceosteoarthritis in the joint of that limb. During week 3 and week 4post-surgery, the mice were treated with 5.8 μg of Nutlin-3A (n=7) peroperated knee by intra-articular injection, q.o.d. for 2 weeks. At theend of 4 weeks post-surgery, joints of the mice were monitored forpresence of senescent cells, assessed for function, monitored formarkers of inflammation, and underwent histological assessment.

Two control groups of mice were included in the studies performed: onegroup comprising C57BL/6J or 3MR mice that had undergone a sham surgery(n=3) (i.e., surgical procedures followed except for cutting the ACL)and intra-articular injections of vehicle parallel to the GCV(ganciclovir) treated group; and one group comprising C57BL/6J or 3MRmice that had undergone an ACL surgery and received intra-articularinjections of vehicle (n=5) parallel to the GCV-treated group. RNA fromthe operated joints of mice from the Nutlin-3A treated mice was analyzedfor expression of SASP factors (mmp3, IL-6) and senescence markers(p16). qRT-PCR was performed to detect mRNA levels.

FIGS. 3A, 3B, and 3C show expression of p16, IL-6, and MMP13 in thetissue, respectively. The OA inducing surgery was associated withincreased expression of these markers. Treatment with Nutlin-3A reducedthe expression back to below the level of the controls. Treatment withNutlin-3A cleared senescent cells from the joint.

Function of the limbs was assessed 4 weeks post-surgery by a weightbearing test to determine which leg the mice favored. The mice wereallowed to acclimate to the chamber on at least three occasions prior totaking measurements. Mice were maneuvered inside the chamber to standwith one hind paw on each scale. The weight that was placed on each hindlimb was measured over a three second period. At least three separatemeasurements were made for each animal at each time point. The resultswere expressed as the percentage of the weight placed on the operatedlimb versus the contralateral unoperated limb.

FIG. 4A shows the results of the functional study. Untreated mice thatunderwent osteoarthritis inducing surgery favored the unoperated hindlimb over the operated hind limb (Δ). However, clearing senescent cellswith Nutlin-3A abrogated this effect in mice that have undergone surgery(∇).

FIGS. 4B, 4C, and 4D show histopathology of joint tissue from theseexperiments. Osteoarthritis induced by ACL surgery caused theproteoglycan layer was destroyed. Clearing of senescent cells usingNutlin-3A completely abrogated this effect.

Example 5: Efficacy of Senolytic Agents in Models for DiabeticRetinopathy

This example illustrates the testing of a Bcl inhibitor in a mouse modelfor treatment of a back-of-the eye disease, specifically diabeticretinopathy. It can be adapted mutatis mutandis to test senolytic agentsfor use in clinical therapy.

The efficacy of model compound UBX1967 (a Bcl-xL inhibitor) was studiedin the mouse oxygen-induced retinopathy (OIR) model (Scott andFruttiger, Eye (2010) 24, 416-421, Oubaha et al, 2016). C57Bl/6 mousepups and their CD1 foster mothers were exposed to a high oxygenenvironment (75% 02) from postnatal day 7 (P7) to P12. At P12, animalswere injected intravitreally with 1 μl test compound (200, 20, or 2 uM)formulated in 1% DMSO, 10% Tween-80, 20% PEG-400, and returned to roomair until P17. Eyes were enucleated at P17 and retinas dissected foreither vascular staining or qRT-PCR. To determine avascular orneovascular area, retinas were flat-mounted, and stained with isolectinB4 (IB4) diluted 1:100 in 1 mM CaCl₂. For quantitative measurement ofsenescence markers (e.g., Cdkn2a, Cdkn1a, 116, Vegfa), qPCR wasperformed. RNA was isolated and cDNA was generated byreverse-transcription, which was used for qRT-PCR of the selectedtranscripts.

FIGS. 5A and 5B show that intravitreal ITT) administration UBX1967resulted in statistically significant improvement in the degree ofneovascularization and vaso-obliteration at all dose levels.

The efficacy of UBX1967 was also studied in the streptozotocin (STZ)model. C57BL/6J mice of 6- to 7-week were weighted and their baselineglycemia was measured (Accu-Chek™, Roche). Mice were injectedintraperitoneally with STZ (Sigma-Alderich, St. Louis, Mo.) for 5consecutive days at 55 mg/Kg. Age-matched controls were injected withbuffer only. Glycemia was measured again a week after the last STZinjection and mice were considered diabetic if their non-fasted glycemiawas higher than 17 mM (300 mg/L). STZ treated diabetic C57BL/6J micewere intravitreally injected with 1 μl of UBX1967 (2 μM or 20 μM,formulated as a suspension in 0.015% polysorbate-80, 0.2% SodiumPhosphate, 0.75% Sodium Chloride, pH 7.2) at 8 and 9 weeks after STZadministration. Retinal Evans blue permeation assay was performed at 10weeks after STZ treatment.

FIGS. 5C and 5D show preliminary results for this protocol. Retinal andchoroidal vascular leakage after intravitreal (IVT) administrationUBX1967 improved in vascular permeability at both dose levels.

Example 6: Efficacy of Senolytic Agents in a Pulmonary Disease Model

This example illustrates the testing of inhibitors in a mouse model fortreatment of lung disease: specifically, a model for idiopathicpulmonary fibrosis (IPF). It can be adapted mutatis mutandis to test anddevelop senolytic agents for use in clinical therapy.

As a model for chronic obstructive pulmonary disease (COPD), mice wereexposed to cigarette smoke. The effect of a senolytic agent on the miceexposed to smoke is assessed by senescent cell clearance, lung function,and histopathology.

The mice used in this study include the 3MR strain, described in US2017/0027139 A1 and in Demaria et al., Dev Cell. 2014 December 22;31(6): 722-733. The 3MR mouse has a transgene encoding thymidine kinasethat converts the prodrug ganciclovir (GCV) to a compound that is lethalto cells. The enzyme in the transgene is placed under control of the p16promoter, which causes it to be specifically expressed in senescentcells. Treatment of the mice with GCV eliminates senescent cells.

Other mice used in this study include the INK-ATTAC strain, described inUS 2015/0296755 A1 and in Baker et al., Nature 2011 Nov. 2;479(7372):232-236. The INK-ATTAC mouse has a transgene encodingswitchable caspase 8 under control of the p16 promoter. The caspase 8can be activated by treating the mice with the switch compound AP20187,whereupon the caspase 8 directly induces apoptosis in senescent cells,eliminating them from the mouse.

To conduct the experiment, six-week-old 3MR (n=35) or INK-ATTAC (n=35)mice were chronically exposed to cigarette smoke generated from a TeagueTE-10 system, an automatically-controlled cigarette smoking machine thatproduces a combination of side-stream and mainstream cigarette smoke ina chamber, which is transported to a collecting and mixing chamber wherevarying amounts of air is mixed with the smoke mixture. The COPDprotocol was adapted from the COPD core facility at Johns HopkinsUniversity (Rangasamy et al., 2004, J. Clin. Invest. 114:1248-1259; Yaoet al., 2012, J. Clin. Invest. 122:2032-2045).

Mice received a total of 6 hours of cigarette smoke exposure per day, 5days a week for 6 months. Each lighted cigarette (3R4F researchcigarettes containing 10.9 mg of total particulate matter (TPM), 9.4 mgof tar, and 0.726 mg of nicotine, and 11.9 mg carbon monoxide percigarette [University of Kentucky, Lexington, Ky.]) was puffed for 2seconds and once every minute for a total of 8 puffs, with the flow rateof 1.05 L/min, to provide a standard puff of 35 cm³. The smoke machinewas adjusted to produce a mixture of side stream smoke (89%) andmainstream smoke (11%) by smoldering 2 cigarettes at one time. The smokechamber atmosphere was monitored for total suspended particulates(80-120 mg/m3) and carbon monoxide (350 ppm).

Beginning at day 7, (10) INK-ATTAC and (10) 3MR mice were treated withAP20187 (3× per week) or ganciclovir (5 consecutive days of treatmentfollowed by 16 days off drug, repeated until the end of the experiment),respectively. An equal number of mice received the correspondingvehicle. The remaining 30 mice (15 INK-ATTAC and 15 3MR) were evenlysplit with 5 of each genetically modified strain placed into threedifferent treatment groups. One group (n=10) received Nutlin-3A (25mg/kg dissolved in 10% DMSO/3% Tween-20™ in PBS, treated 14 daysconsecutively followed by 14 days off drug, repeated until the end ofthe experiment). One group (n=10) received ABT-263 (Navitoclax) (100mg/kg dissolved in 15% DMSO/5% Tween-20, treated 7 days consecutivelyfollowed by 14 days off drug, repeated until the end of the experiment),and the last group (n=10) received only the vehicle used for ABT-263(15% DMSO/5% Tween-20), following the same treatment regimen as ABT-263.An additional 70 animals that did not receive exposure to cigarettesmoke were used as controls for the experiment.

After two months of cigarette smoke (CS) exposure, lung function wasassessed by monitoring oxygen saturation using the MouseSTATPhysioSuite™ pulse oximeter (Kent Scientific). Animals were anesthetizedwith isoflurane (1.5%) and the toe clip was applied. Mice were monitoredfor 30 seconds and the average peripheral capillary oxygen saturation(SpO2) measurement over this duration was calculated.

FIG. 6 shows the results. Clearance of senescent cells via AP2018,ganciclovir, ABT-263 (Navitoclax) (201), or Nutlin-3A (101) resulted instatistically significant increases in SpO₂ levels in mice after twomonths of cigarette smoke exposure, compared with untreated controls.

Example 7: Efficacy of Senolytic Agents in Atherosclerosis whenAdministered Systemically

This example illustrates the testing of an MDM2 inhibitor in a mousemodel for treatment of atherosclerosis. The test compounds areadministered systemically rather than locally. The model is done in anLDLR−/− strain of mice, which are deficient in the receptor forlow-density lipoprotein. The experiments described here can be adaptedmutatis mutandis to test and develop other types of inhibitors for usein clinical therapy.

Two groups of LDLR−/− mice (10 weeks) are fed a high fat diet (HFD)(Harlan Teklad TD.88137) having 42% calories from fat, beginning at Week0 and throughout the study. Two groups of LDLR−/− mice (10 weeks) arefed normal chow (−HFD). From weeks 0-2, one group of HFD mice and −HFDmice are treated with Nutlin-3A (25 mg/kg, intraperitoneally). Onetreatment cycle is 14 days treatment, 14 days off. Vehicle isadministered to one group of HFD mice and one group of −HFD mice. Atweek 4 (timepoint 1), one group of mice are sacrificed and to assesspresence of senescent cells in the plaques. For the some of theremaining mice, Nutlin-3A and vehicle administration is repeated fromweeks 4-6. At week 8 (timepoint 2), the mice are sacrificed and toassess presence of senescent cells in the plaques. The remaining miceare treated with Nutlin-3A or vehicle from weeks 8-10. At week 12(timepoint 3), the mice are sacrificed and to assess the level of plaqueand the number of senescent cells in the plaques.

Plasma lipid levels were measured in LDLR−/− mice fed a HFD and treatedwith Nutlin-3A or vehicle at timepoint 1 as compared with mice fed a−HFD (n=3 per group). Plasma was collected mid-afternoon and analyzedfor circulating lipids and lipoproteins.

At the end of timepoint 1, LDLR−/− mice fed a HFD and treated withNutlin-3A or vehicle were sacrificed (n=3, all groups), and the aorticarches were dissected for RT-PCR analysis of SASP factors and senescentcell markers. Values were normalized to GAPDH and expressed asfold-change versus age-matched, vehicle-treated LDLR−/− mice on a normaldiet. The data show that clearance of senescent cells with Nutlin-3A inLDLR−/− mice fed a HFD reduced expression of several SASP factors andsenescent cell markers, MMP3, MMP13, PAI1, p21, IGFBP2, IL-1A, and IL-1Bafter one treatment cycle.

At the end of timepoint 2, LDLR−/− mice fed a HFD and treated withNutlin-3A or vehicle (n=3 for all groups) were sacrificed, and aorticarches were dissected for RT-PCR analysis of SASP factors and senescentcell markers. Values were normalized to GAPDH and expressed asfold-change versus age-matched, vehicle-treated LDLR−/− mice on a normaldiet. The data show expression of some SASP factors and senescent cellmarkers in the aortic arch within HFD mice. Clearance of senescent cellswith multiple treatment cycles of Nutlin-3A in LDLR−/− mice fed a HFDreduced expression of most markers.

At the end of timepoint 3, LDLR−/− mice fed a HFD and treated withNutlin-3A or vehicle (n=3 for all groups) were sacrificed, and aortaswere dissected and stained with Sudan IV to detect the presence oflipid. Body composition of the mice was analyzed by MRI, and circulatingblood cells were counted by Hemavet™.

FIG. 7 shows the results. Treatment with Nutlin-3A reduced the surfacearea covered by plaques in the descending aorta by about 45%. Theplatelet and lymphocyte counts were equivalent between the Nutlin-3A andvehicle treated mice. Treatment with Nutlin-3A also decreased mass andbody fat composition in mice fed the high fat diet.

Example 8: Measuring Cytotoxicity for Cancer Cells In Vitro and In Vivo

New proteasome inhibitors may be developed not only for treatingconditions mediated by senescent cells, but also conditions mediated bycancer cells.

The ability of compounds to specifically kill cancer cells can be testedin assays using other established cell lines. These include HeLa cells,OVCAR-3, LNCaP, and any of the Authenticated Cancer Cell Lines availablefrom Millipore Sigma, Burlington Mass., U.S.A. Compounds specificallykill cancer cells if they are lethal to the cells at a concentrationthat is at least 5-fold lower, and preferably 25- or 100-fold lower thana non-cancerous cell of the same tissue type. The control cell hasmorphologic features and cell surface markers similar to the cancer cellline being tested, but without signs of cancer.

In vivo, compounds are evaluated in flank xenograft models establishedfrom sensitive SCLC (H889) and hematologic (RS4;11) cell lines, or usingother tumor-forming cancer cell lines, according to what type of canceris of particular interest to the user. When dosed orally orintravenously, compounds induce rapid and complete tumor responses (CR)that are durable for several weeks after the end of treatment in allanimals bearing H889 (SCLC) or RS4;11 (ALL) tumors. Similar treatment ofmice bearing H146 SCLC tumors can induce rapid regressions in theanimals.

EXEMPLARY COMPOUNDS AND THEIR USE

Some of the proteasome inhibitors that illustrate this invention andtheir use are set forth in the following clauses.

Clause 1. A compound according to Formula (I) that inhibits proteasomeactivity:R⁰—X-(A)_(n)-Z  (I)

wherein;

-   -   R⁰ is selected from H, alkyl, substituted alkyl, alkanoyl,        substituted alkanoyl, alkylaminocarbonyl, substituted        alkylaminocarbonyl, alkoxycarbonyl, substituted alkoxycarbonyl,        alkylaminothiocarbonyl, substituted alkylaminothiocarbonyl,        alkoxythiocarbonyl, substituted alkoxythiocarbonyl and        promoiety;    -   (A)^(n) is a sequence of n peptidic units A¹ to A^(n), each        independently selected from amino acid residues and        peptidomimetic units, wherein:        -   n is 2-7;        -   the A¹ unit comprises a first terminal group X, wherein X is            O or S; and        -   the A^(n) unit comprises a second terminal group Z; and

Z is a proteasome-reactive electrophilic group.

Clause 2. The proteasome inhibitor compound of clause 1, wherein thecompound is of Formula (Ia):R⁰—X-A¹-A²-A³-A⁴-Z  (Ia)

wherein:

-   -   n is 4; and    -   A¹ to A⁴ are independently selected from amino acid residues        (e.g., α-amino acids, β-amino acids, D-amino acids) and        peptidomimetic units (for example, retroinverso peptide units,        peptoid units, hydroxyethylamine isosteric units, as described        herein), wherein the A⁴ unit comprises a modified terminal        comprising Z.

Clause 3. The proteasome inhibitor compound of clause 1 or 2, whereinthe compound is of Formula (II):

wherein:

-   -   B¹ to B⁴ are each independently a branching group selected from        CH, CR″ and N, wherein R″ is C₍₁₋₆₎alkyl or substituted        C₍₁₋₆₎alkyl;    -   L¹ to L⁴ are each independently a linking group (e.g., a group        having a backbone of 1-3 atoms); and    -   R¹ to R⁴ are independently selected from alkyl, substituted        alkyl, aralkyl, substituted aralkyl, heteroarylalkyl and        substituted heteroarylalkyl.

Clause 4. The proteasome inhibitor compound of clause 3, wherein L¹ toL⁴ are each independently selected from —CONR′—, —CH₂NR′—, CH(OH)NR′—,—CH₂CONR′—, —CO—, —CH₂CO—, —COCH₂—, —CO₂—, —CH₂O—, —CH₂S—, —CH₂CH₂—,—CH═CH— and —NR′CO—, wherein R′ is H, C₍₁₋₆₎alkyl or substitutedC₍₁₋₆₎alkyl (e.g., methyl).

Clause 5. The proteasome inhibitor compound of clause 3 or 4, wherein atleast one —B^(n)(R^(n))-L^(n)— group of formula (II) is selected fromthe following groups:

Clause 6. The proteasome inhibitor compound of any one of clauses 3-5,wherein the compound is of one of Formulas (IIIa) to (IIIc):

Clause 7. The proteasome inhibitor compound of any one of clauses 1-6,wherein Z is selected from aldehyde, epoxyketone, aziridinylketone,boronate, boronate ester and beta-lactone.

Clause 8. The proteasome inhibitor compound of any one of clauses 3-7,wherein R¹ to R⁴ are selected from one of the following combinations#1-6:

# R¹ R² R³ R⁴ 1 phenyl-C₍₁₋₆₎alkyl, CH₂CH(CH₃)₂ CH₂Ph CH₂CH(CH₃)₂cycloalkyl-C₍₁₋₆₎alkyl or C₍₁₋₆₎alkyl 2 CH₂Ph C₍₁₋₆₎alkoxy-C₍₁₋₆₎alkyl,CH₂Ph CH₂CH(CH₃)₂ C₍₁₋₆₎alkyl or C₍₁₋₆₎hydroxyalkyl 3 CH₂CH₂PhCH₂CH(CH₃)₂ CH₂CH(CH₃)₂ 4 CH₂CH₂Ph CH₂CH(CH₃)₂ CH₂Ph C₍₁₋₆₎alkyl 5CH₂CH₂Ph C₍₁₋₆₎alkoxy-C₍₁₋₆₎alkyl, CH₂Ph C₍₁₋₆₎alkyl C₍₁₋₆₎alkyl orC₍₁₋₆₎hydroxyalkyl 6 CH₂CH₂Ph CH₂CH(CH₃)₂ CH₂Ph CH₂CH(CH₃)₂

Clause 9. The proteasome inhibitor compound of any of clauses 1 to 8,wherein:

-   -   X is O or S;    -   R⁰- is R¹⁰- or R¹⁰-Q-;    -   Q is selected from ethylene glycol, polyethylene glycol,        —C(═O)—, —NR¹C(═O)—, —OC(═O)—, —C(═S)—, —NR¹C(═S)—, —OC(═S)— and        —OC(═S)—; and    -   R¹⁰ and R¹¹ are independently selected from H, alkyl and        substituted alkyl.

Clause 10. The proteasome inhibitor compound of clause 9, wherein R⁰ isselected from the following structures:

wherein:

-   -   m is an integer from 1 to 6;    -   p is an integer from 1 to 30;    -   X′ is selected from O, S and NR¹⁴; and    -   R¹² and R¹³ are independently selected from H, alkyl and        substituted alkyl, or R¹² and R¹³ are cyclically linked and        together with the nitrogen atom to which they are attached        provide a heterocycle ring that is optionally further        substituted; and    -   each R¹⁴ is independently selected from H, C₍₁₋₆₎alkyl and        substituted C₍₁₋₆₎alkyl.

Clause 11. A compound according to Formula (IV) that inhibits proteasomeactivity:

wherein:

-   -   R² to R⁴ are independently selected from alkyl, substituted        alkyl, aralkyl, substituted aralkyl, heteroarylalkyl and        substituted heteroarylalkyl; and    -   Z¹ is epoxide group or aziridine group.

Clause 12. The proteasome inhibitor compound of clause 11, having thestructure shown in Formula (V):

wherein:

-   -   Y is selected from O and NR¹⁵; and    -   R⁵ and R¹⁵ are independently selected from H, C₍₁₋₆₎alkyl and        substituted C₍₁₋₆₎alkyl.

Clause 13. The proteasome inhibitor compound of any one of clauses 11 to12, wherein R¹ to R⁴ are independently selected from C₍₁₋₆₎alkyl,substituted C₍₁₋₆₎alkyl, C₍₁₋₆₎hydroxyalkyl, substitutedC₍₁₋₆₎hydroxyalkyl, C₍₁₋₆₎alkoxy-C₍₁₋₆₎alkyl, substitutedC₍₁₋₆₎alkoxy-C₍₁₋₆₎alkyl, aryl-C₍₁₋₆₎alkyl, substitutedaryl-C₍₁₋₆₎alkyl, heteroaryl-C₍₁₋₆₎alkyl, substitutedheteroaryl-C₍₁₋₆₎alkyl, cycloalkyl-C₍₁₋₆₎alkyl, substitutedcycloalkyl-C₍₁₋₆₎alkyl, heterocycle-C₍₁₋₆₎alkyl and substitutedheterocycle-C₍₁₋₆₎alkyl.

Clause 14. The proteasome inhibitor compound of any one of clauses 11 to12, wherein:

-   -   R¹ is selected from C₍₁₋₆₎alkyl, aryl-C₍₁₋₆₎alkyl, substituted        aryl-C₍₁₋₆₎alkyl, cycloalkyl-C₍₁₋₆₎alkyl and substituted        cycloalkyl-C₍₁₋₆₎alkyl;    -   R² and R⁴ are selected from C₍₁₋₆₎alkyl, substituted        C₍₁₋₆₎alkyl, C₍₁₋₆₎alkoxy-C₍₁₋₆₎alkyl, substituted        C₍₁₋₆₎alkoxy-C₍₁₋₆₎alkyl, C₍₁₋₆₎hydroxyalkyl and substituted        C₍₁₋₆₎hydroxyalkyl; and    -   R³ is selected from aryl-C₍₁₋₆₎alkyl, substituted        aryl-C₍₁₋₆₎alkyl, C₍₁₋₆₎alkoxy-C₍₁₋₆₎alkyl and substituted        C₍₁₋₆₎alkoxy-C₍₁₋₆₎alkyl.

Clause 15. The proteasome inhibitor compound of any one of clauses 11 to12, wherein:

-   -   R¹ is selected from phenyl-C₍₁₋₆₎alkyl, cycloalkyl-C₍₁₋₆₎alkyl        and C₍₁₋₆₎alkyl;    -   R² is selected from C₍₁₋₆₎alkoxy-C₍₁₋₆₎alkyl, C₍₁₋₆₎alkyl and        C₍₁₋₆₎hydroxyalkyl;    -   R³ is selected from phenyl-C₍₁₋₆₎alkyl, cycloalkyl-C₍₁₋₆₎alkyl,        C₍₁₋₆₎alkoxy-C₍₁₋₆₎alkyl and C₍₁₋₆₎hydroxyalkyl; and    -   R⁴ is C₍₁₋₆₎alkyl.

Clause 16. The proteasome inhibitor compound of any one of clauses 11 to12, wherein:

-   -   R¹ is selected from phenylethyl, cyclopropyl-methyl and propyl;    -   R² is selected from methoxymethyl, isobutyl and 1-hydroxy-ethyl;    -   R³ is selected from phenylmethyl, cyclopropyl-methyl,        methoxymethyl and 1-hydroxy-ethyl; and    -   R⁴ is isobutyl.

Clause 17. The proteasome inhibitor compound of any of clauses 11 to 16,having the stereochemistry shown in Formula (VI):

Clause 18. The proteasome inhibitor compound of clause 17, selected fromthe following Formulas (VIa)-(VId):

Clause 19. The proteasome inhibitor compound of clause 17, having thestructure shown in Formula (VIe):

Clause 20. The proteasome inhibitor compound of clause 17, having thestructure shown in Formula (VIf):

Clause 21. The proteasome inhibitor compound of any of clauses 17 to 20,wherein Y is O and R⁵ is methyl.

Clause 22. The proteasome inhibitor compound of any of clauses 17 to 20,wherein X is O or S.

Clause 23. The proteasome inhibitor compound of any of clauses 11 to 22,wherein:

-   -   X is O or S;    -   R⁰- is R¹⁰- or R¹⁰-Q-;    -   Q is selected from ethylene glycol, polyethylene glycol,        —C(═O)—, —NR“C(═O)—, —OC(═O)—, —C(═S)—, —NR” C(═S)—, —OC(═S)—        and —OC(═S)—; and    -   R¹⁰ and R¹¹ are independently selected from H, alkyl and        substituted alkyl.

Clause 24. The proteasome inhibitor compound of clause 23, wherein R⁰ isselected from the following structures:

wherein:

-   -   m is an integer from 1 to 6;    -   p is an integer from 1 to 30;    -   X¹ is selected from O, S and NR¹⁴; and    -   R¹² and R¹³ are independently selected from H, alkyl and        substituted alkyl, or R¹² and R¹³ are cyclically linked and        together with the nitrogen atom to which they are attached        provide a heterocycle ring that is optionally further        substituted; and    -   each R¹⁴ is independently selected from H, C₍₁₋₆₎alkyl and        substituted C₍₁₋₆₎alkyl.

Clause 25. The proteasome inhibitor compound of clause 23, wherein R⁰ isselected from the following structures:

wherein:

-   -   q is an integer from 1 to 3 (e.g., q is 3);    -   Y² is selected from O and NR¹⁵ (e.g., Y² is O); and    -   R¹⁵ is selected from H, C₍₁₋₆₎alkyl and substituted C₍₁₋₆₎alkyl.

Clause 26. The proteasome inhibitor compound of clause 25, wherein q is3; and Y² is O.

Clause 27. The proteasome inhibitor compound according to any of clauses1 to 26, which has an EC₅₀ for irradiated IMR90 cells of less than 0.1μM (100 nM).

Clause 28. The proteasome inhibitor compound according to any of clauses1 to 27, which has a EC₅₀ for senescent cells that is at least 10-foldhigher than its EC₅₀ for non-senescent cells of the same cell type.

Clause 30. The proteasome inhibitor compound of clause 1, selected fromthe compounds shown in FIGS. 1A and 1B.

Clause 31. A proteasome inhibitor according to any of clauses 1 to 30,for use in the selective removal of senescent cells from a mixed cellpopulation or tissue, or for use in treatment of a senescence associatedcondition that is caused or mediated at least in part by senescentcells.

Clause 32. A proteasome inhibitor according to any of clauses 1 to 30,for use in the selective removal of cancer cells, or for use intreatment of cancer.

Clause 33. A method of selectively removing senescent cells from a cellpopulation or tissue, comprising selectively inhibiting activity ofproteasome in the cell.

Clause 34. A method of modulating or eliminating a senescent cell from acell population or tissue, comprising contacting the senescent cell witha means for inhibiting activity of proteasome.

Clause 35. The method of clause 33 or 34, wherein the proteasomeinhibitor is a compound according to any of clauses 1 to 30.

Clause 36. The method of clause 33 or 34, wherein the proteasomeinhibitor is a compound selected from the structures shown in FIGS. 1A,1B, and 1C.

Clause 37. A method of treating a senescence related condition in atissue in a subject,

-   -   wherein the senescence related condition a condition that is        caused or mediated at least in part by senescent cells in the        tissue, or is characterized as having an overabundance of        senescent cells in or around the tissue, in comparison with        unaffected tissue,    -   wherein the method comprises administering to the tissue an        effective amount of a means for inhibiting activity of        proteasome, thereby selectively removing senescent cells from        the tissue and relieving at least one sign or symptom of the        condition in the subject.

Clause 38. A unit dose of a pharmaceutical composition that contains anamount of a compound that inhibits activity of proteasome, configuredfor use in the treatment of a senescence associated condition that iscaused or mediated at least in part by senescent cells,

-   -   wherein the composition contains a formulation of the compound        configured for administration to a tissue in a subject that        manifests the condition,    -   wherein the formulation of the composition and the amount of the        compound in the unit dose configure the unit dose to be        effective in selectively removing senescent cells in or around        the tissue in the subject, thereby decreasing the severity of        one or more signs or symptoms of the condition without causing        adverse effects in the subject when administered to the tissue        as a single dose.

Clause 39. The product or method of clause 37 or 38, wherein theproteasome inhibitor is an inhibitor according to any of clauses 1 to30.

Clause 40. The product or method of clause 37 or 38, wherein theproteasome inhibitor is an inhibitor selected from the structures shownin FIGS. 1A, 1B, and 1C.

Clause 41. The product or method of any of clauses 37 to 40, wherein thecondition is osteoarthritis.

Clause 42. The product or method of any of clauses 37 to 40, wherein thecondition is an ophthalmic condition.

Clause 43. The product or method of clause 42, wherein the ophthalmiccondition is selected from wet and dry age-related macular degeneration(AMD), diabetic retinopathy, and glaucoma.

Clause 44. The product or method of any of clauses 37 to 40, wherein thecondition is a pulmonary condition.

Clause 45. The product or method of clause 44, wherein the pulmonarycondition is selected from chronic obstructive pulmonary disease (COPD)and idiopathic pulmonary fibrosis (IPF).

Clause 46. The product or method of any of clauses 37 to 40, wherein thecondition is atherosclerosis.

The several hypotheses presented in this disclosure provide a premise byway of which the reader may understand the invention. This premise isprovided for the intellectual enrichment of the reader. Practice of theinvention does not require detailed understanding or application of thehypothesis. Except where stated otherwise, features of the hypothesispresented in this disclosure do not limit application or practice of theclaimed invention.

For example, except where the elimination of senescent cells isexplicitly required, the compounds may be used for treating theconditions described regardless of their effect on senescent cells.Although many of the senescence-related conditions referred to in thisdisclosure occur predominantly in older patients, the occurrence ofsenescent cells and the pathophysiology they mediate can result fromother events, such as irradiation, other types of tissue damage, othertypes of disease, genetic abnormalities, and invention. The inventionmay be practiced on patients of any age having the condition indicated,unless otherwise explicitly indicated or required.

Discussions about the mechanism of action of the peptide-based compoundsof the invention are also provided for the intellectual enrichment ofthe reader. Except where stated otherwise, the compounds may be used forremoving senescent or cancer cells or for the treatment of diseaseconditions as claimed below, regardless of how they operate inside thetarget cells or in the treated subject.

Although the compounds and compositions referred to in this disclosureare illustrated in the context of eliminating senescent cells andtreating senescence-associated conditions, compounds and theirderivatives described herein that are novel can be prepared for anypurpose, including but not limited to laboratory use, the treatment ofsenescence-related conditions, the poisoning of in-laws, and thetreatment of other conditions such as cancer.

While the invention has been described with reference to the specificexamples and illustrations, changes can be made and equivalents can besubstituted to adapt to a particular context or intended use as a matterof routine development and optimization and within the purview of one ofordinary skill in the art, thereby achieving benefits of the inventionwithout departing from the scope of what is claimed and theirequivalents.

The invention claimed is:
 1. A method for selectively removing a senescent cell from a mixed cell population or tissue by inhibiting proteasome activity in the cell, comprising contacting the cell with a compound according to Formula (V):

wherein: R⁰ is selected from H, alkyl, substituted alkyl, alkanoyl, substituted alkanoyl, alkylaminocarbonyl, substituted alkylaminocarbonyl, alkoxycarbonyl, substituted alkoxycarbonyl, alkylaminothiocarbonyl, substituted alkylaminothiocarbonyl, alkoxythiocarbonyl, substituted alkoxythiocarbonyl and promoiety; X is O or S; R¹ to R⁴ are independently selected from alkyl, substituted alkyl, aralkyl, substituted aralkyl, heteroarylalkyl and substituted heteroarylalkyl; Y is selected from O and NR¹⁵; and R⁵ and R¹⁵ are independently selected from H, C₍₁₋₆₎alkyl and substituted C₍₁₋₆₎alkyl.
 2. The method of claim 1, wherein: R¹ is selected from C₍₁₋₆₎alkyl, aryl-C₍₁₋₆₎alkyl, substituted aryl-C₍₁₋₆₎alkyl, cycloalkyl-C₍₁₋₆₎alkyl and substituted cycloalkyl-C₍₁₋₆₎alkyl; R² and R⁴ are selected from C₍₁₋₆₎alkyl, substituted C₍₁₋₆₎alkyl, C₍₁₋₆₎alkoxy-C₍₁₋₆₎alkyl, substituted C₍₁₋₆₎alkoxy-C₍₁₋₆₎alkyl, C₍₁₋₆₎hydroxyalkyl and substituted C₍₁₋₆₎hydroxyalkyl; and R³ is selected from aryl-C₍₁₋₆₎alkyl, substituted aryl-C₍₁₋₆₎alkyl, C₍₁₋₆₎alkoxy-C₍₁₋₆₎alkyl and substituted C₍₁₋₆₎alkoxy-C₍₁₋₆₎alkyl.
 3. The method of claim 1, wherein; R¹ is selected from phenylethyl, cyclopropyl-methyl and propyl; R² is selected from methoxymethyl, isobutyl and 1-hydroxy-ethyl; R³ is selected from phenylmethyl, cyclopropyl-methyl, methoxymethyl and 1-hydroxy-ethyl; and R⁴ is isobutyl.
 4. The method of claim 1, wherein the compound is selected from the following:


5. The method of claim 4, wherein: Y is O, R⁵ is methyl; and X is O.
 6. The method of claim 1, wherein: X is O or S; R⁰- is R¹⁰- or R¹⁰-Q-; Q is selected from ethylene glycol, polyethylene glycol, —C(═O)—, —NR¹¹C(═O)—, —OC(═O)—, —C(═S)—, —NR11C(═S)—, —OC(═S)— and —OC(═S)—; and R¹⁰ and R¹¹ are independently selected from H, alkyl and substituted alkyl.
 7. A method for selectively removing a cancer cell from a mixed cell population or tissue by inhibiting proteasome activity in the cell comprising contacting the cell with a compound according to Formula (IV):

wherein: R⁰ is selected from H, alkyl, substituted alkyl, alkanoyl, substituted alkanoyl, alkylaminocarbonyl, substituted alkylaminocarbonyl, alkoxycarbonyl, substituted alkoxycarbonyl, alkylaminothiocarbonyl, substituted alkylaminothiocarbonyl, alkoxythiocarbonyl, substituted alkoxythiocarbonyl and promoiety; X is O or S; R¹ to R⁴ are independently selected from alkyl, substituted alkyl, aralkyl, substituted aralkyl, heteroarylalkyl and substituted heteroarylalkyl; Y is selected from O and NR¹⁵; and R⁵ and R¹⁵ are independently selected from H, C₍₁₋₆₎alkyl and substituted C₍₁₋₆₎alkyl.
 8. The method of claim 7, wherein: R¹ is selected from C₍₁₋₆₎alkyl, aryl-C₍₁₋₆₎alkyl, substituted aryl-C₍₁₋₆₎alkyl, cycloalkyl-C₍₁₋₆₎alkyl and substituted cycloalkyl-C₍₁₋₆₎alkyl; R² and R⁴ are selected from C₍₁₋₆₎alkyl, substituted C₍₁₋₆₎alkyl, C₍₁₋₆₎alkoxy-C₍₁₋₆₎alkyl, substituted C₍₁₋₆₎alkoxy-C₍₁₋₆₎alkyl, C₍₁₋₆₎hydroxyalkyl and substituted C₍₁₋₆₎hydroxyalkyl; and R³ is selected from aryl-C₍₁₋₆₎alkyl, substituted aryl-C₍₁₋₆₎alkyl, C₍₁₋₆₎alkoxy-C₍₁₋₆₎alkyl and substituted C₍₁₋₆₎alkoxy-C₍₁₋₆₎alkyl.
 9. The method of claim 7, wherein; R¹ is selected from phenylethyl, cyclopropyl-methyl and propyl; R² is selected from methoxymethyl, isobutyl and 1-hydroxy-ethyl; R³ is selected from phenylmethyl, cyclopropyl-methyl, methoxymethyl and 1-hydroxy-ethyl; and R⁴ is isobutyl.
 10. The method of claim 7, wherein the compound is selected from the following:


11. The method of claim 10, wherein: Y is O, R⁵ is methyl; and X is O.
 12. The method of claim 7, wherein: X is O or S R⁰- is R¹⁰- or R¹⁰-Q-; Q is selected from ethylene glycol polyethylene glycol, —C(═O)—, —NR¹¹C(═O)—, —OC(═O)—, —C(═S)—, —NR11C(═S)—, —OC(═S)— and —OC(═S)—; and R¹⁰ and R¹¹ are independently selected from H, alkyl and substituted alkyl.
 13. A method for selectively removing a senescent cell from a mixed cell population or tissue, comprising contacting the cell with a compound selected from the following:


14. The method of claim 7, wherein the compound is selected from the following: 